JP7157045B2 - Circulating ESM-1 (endocan) in the assessment of atrial fibrillation and stroke - Google Patents

Description

The present invention is a method of assessing atrial fibrillation in a subject comprising the steps of determining the amount of ESM-1 in a sample from said subject and comparing said amount of ESM-1 to a reference amount thereby evaluating movement. Further, the present invention relates to a method of diagnosing heart failure and/or at least one cardiac structural or functional abnormality associated with heart failure.

Atrial fibrillation is the most common type of cardiac arrhythmia and one of the most prevalent conditions in the aging population. Atrial fibrillation is characterized by irregular heartbeats, often beginning with short periods of abnormal heartbeats that increase over time and can become a permanent condition. Atrial fibrillation (AF) affects an estimated 2.7–6.1 million people in the United States and approximately 33 million people worldwide (Chugh S.S. et al., Circulation 2014; 129: 837-47). ). AF patients have a high incidence of stroke and are at increased risk of developing congestive heart failure compared to patients in sinus rhythm.

Diagnosis of cardiac arrhythmias, such as atrial fibrillation, typically involves determining the cause of the arrhythmia and classifying the arrhythmia. Guidelines for the classification of atrial fibrillation according to the American College of Cardiology (ACC), American Heart Association (AHA), and European Society of Cardiology (ESC) are primarily simplified. based on sex and clinical relevance. The first category is called "incipient AF". Individuals in this category were initially diagnosed with AF and either already had an undetected episode (episode) or not. If the onset of the first symptom ceases spontaneously within 1 week, but another symptom develops thereafter, the category is moved to "paroxysmal AF." Patients in this category have episodes lasting up to 7 days, and the majority of paroxysmal AF will cease onset within 24 hours. If an episode lasts for more than a week, it is classified as "persistent AF". If such episodes cannot be stopped, ie, cannot be stopped by electrical or pharmacological defibrillation, and persist for more than a year, the classification changes to "permanent AF." Because atrial fibrillation is a significant risk factor for stroke and systemic embolism, early diagnosis of atrial fibrillation is highly desirable (Hart et al., Ann. Intern Med 2007; 146(12): 857-67; Go AS et al. JAMA 2001; 285(18):2370-5).

ESM-1 (also known as endocan) is a proteoglycan composed of a 20 kDa mature polypeptide and 30 kDa O-linked glycan chains (Bechard D. et al., J Biol Chem 2001; 276(51) : 48341-48349). The carboxylates and sulfates of the glycan chains are negatively charged at physiological pH and thus provide binding sites for signaling molecules containing positively charged amino acids, such as growth factors and cytokines (Roudnicky F. et al., Cancer Res. 2013;73:(3):1097-106). Biological ESM-1 expression and release from endothelial cells is highly induced by the angiogenic mediators VEGF-A, VEGF-C, FGF-2, PI3K and cytokines involved in cancer progression is also induced by inflammatory processes (sepsis) (Kali A. et al.; Indian J. Pharmacol. 2014 46(6): 579-583). ESM-1 binds and upregulates pro-angiogenic growth factors such as FGF-2 and HGF, thereby mediating increased migration and proliferation of endothelial cells. Endocan mutants lacking glycan chains were unable to induce HGF activation, highlighting the role of glycans (Delehedde M. et al.; Int J Cell Biol. 2013:705027). ESM-1 binds to the LFA-1 integrin (CD11a/CD18) on the cell surface of blood lymphocytes, monocytes, and Jurkat cells and recruits blood lymphocytes to sites of inflammation, resulting in LFA-1 dependence. induces sexual leukocyte adhesion and activation.

Soluble ESM-1 was discovered as a risk factor marker for endothelial dysfunction in various cancer types. Additionally, the markers have been evaluated in association with various cardiovascular conditions or diseases. For example, ESM-1 is associated with hypertension (Balta S. et al. Angiology. 2014; 65(9):773-7), coronary artery disease and myocardial infarction (Kose M. et al. Angiology. 2015, 66(8): 727-31 ). Furthermore, the marker was also measured in diabetic patients (Arman Y. et al., Angiology. 2016 Mar; 67(3):239-44). Measurements at different stages of chronic kidney disease led to the conclusion that this marker could also be useful in inferring cardiovascular events and mortality in chronic kidney disease (Yilmaz MI. et al., Kidney Int. 2014). 86(6):1213-20).

Mosevoll et al. (Springerplus. 2014 Sep 30; 3:571) analyzed endocan and E-selectin in patients with suspected deep vein thrombosis. Plasma endocan and E-selectin levels were measured in patients with thrombosis, healthy controls, patients without documented thrombosis (i.e. patients with their symptoms of other causes, e.g. patients with various inflammatory and non-inflammatory conditions). There was no difference between However, the combination of endogenous biomarkers C-reactive protein and D-dimer could be used to identify patient subsets with different frequencies of venous thrombosis. Increased endocan levels in patients with extensive pulmonary embolism were due to embolism rather than primary thrombosis and/or malignant circulation with pulmonary hypertension/right heart failure. The authors concluded that "increased endocan levels in patients with extensive pulmonary embolism are due to embolism rather than primary thrombosis and/or malignant circulation with pulmonary hypertension/right heart failure" (Mosevoll et al. et al., SpringerPlus 2014, 3:571).

Furthermore, endocan release into blood is considered to be a biomarker of endothelial dysfunction, changes in vascular permeability and severity of sepsis (Chelazzi C. et al.; Crit Care. 2015; 19(1): 26). It has also been shown that endocans are expressed during the angiogenic process following the inflammatory process (Matano F. et al., J Neurooncol. 2014 May, 117(3): 485-91).

A conference abstract from Menon et al. describes the effects of ESM-1 knockout in a mouse model. The authors observed a dilated aorta. Furthermore, upregulation of nppa, nppb and myh7 in knockout hearts was also observed. The authors concluded that disruption of ESM-1 can cause cardiac dysfunction (Abstract 19140: Targeted Disruption of Endothelial Specific Molecule (ESM)-1 Results in Atrioventricular Valve Malformations Leading to Cardiac Dysfunction. Prashanthi Menon, Katherine Spokes , David Beeler, Lauren Janes, and William Aird Circulation. 2012; 126: A19140 Vol. 126, No. 21 Supplement;

Xion et al. measured endocan levels in hypertensive patients. The marker was described to be elevated in the hypertensive group versus the non-hypertensive group. Among the hypertensive group, patients with coronary artery disease CAD had higher levels than those without CAD (Xiong C. et al., Elevated Human Endothelia Cell-Specific Molecule-1 Level and Its Association With Coronary Artery Disease in Patients With Hypertension. J Investig Med. 2015 Oct;63(7):867-70).

Qiu et al. measured ESM-1 in type II diabetic patients with acute STEMI myocardial infarction (Qiu CR. et al., Angiology. 2017 Jan;68(1):74-78). The authors describe a difference in serum ESM-1 levels between a type II diabetic group with STEMI (ST-segment elevation myocardial infarction) and a type II diabetic group without vascular disease. Determination of endothelial dysfunction predicts cardiovascular risk, such as myocardial infarction.

Cimen et al. studied the relationship between obstructive coronary artery disease (CAD) and microvascular angina (MVA) and endocan plasma levels (Cimen T. et al., Angiology. 2016; 67(9): 846-853). . For example, patients with atrial fibrillation were not analyzed. CAD patients with microvascular angina (MVA) showed increased ESM-1 compared with CAD controls. The authors proposed that the marker could serve as an indicator of endothelial dysfunction prior to overt cardiovascular disease.

Madhivathanan et al. analyzed the pharmacokinetics of endocan in cardiac surgical patients with acute lung injury as a complication of cardiac surgery (bypass surgery as well as complex surgery). The authors concluded that baseline endocan concentrations were higher in hypertensive patients with severe coronary stenosis, and that endocan concentrations would increase after induction of anesthesia but decrease 4 hours after separation from CPB (Madhivathanan PR et al. , Cytokine. 2016 Jul; 83:8-12).

WO1999/045028 describes two specific monoclonal antibodies for detecting ESM-1.

WO2002/039123 discloses a kit for detecting ESM-1 protein and for in vitro quantification of ESM-1 protein in patients treated with immunosuppressive compounds and ESM-1 protein in patients suffering from cancer. describes the use of ESM-1 to detect in vitro deterioration of human endothelial vessel walls for in vitro quantification of 1.

WO2012/098219 describes ESM-1 as a marker for predicting the risk of respiratory failure, renal failure and thrombocytopenia in patients with sepsis.

WO2014/135488 describes ESM-1 as a marker for identifying pregnancy-related syndromes such as pre-eclampsia and IUGR.

Latini R. et al. (J Intern Med. 2011 Feb; 269(2): 160-71) reported that various circulating biomarkers (hsTnT, NT-proBNP, MR-proANP, MR-proADM , copeptin and CT-proendothelin-1) were measured.

Atrial fibrillation management, including diagnosis of atrial fibrillation, risk stratification of patients with atrial fibrillation (e.g., stroke incidence), assessment of severity of atrial fibrillation and assessment of treatment for patients with atrial fibrillation A valuation method is needed.

It can be understood that the technical problem underlying the present invention is to provide a method for meeting the above needs. This technical problem is solved by the embodiments characterized in the claims and described below.

Advantageously, it was found in the study of the present invention that determination of ESM-1 amount in a sample from a subject allows an improved assessment of atrial fibrillation. The present invention makes it possible, for example, to diagnose whether a subject is suffering from atrial fibrillation. The present invention also provides a method of stroke incidence prediction. Furthermore, the present invention can distinguish between paroxysmal and persistent atrial fibrillation in patients with atrial fibrillation.

Accordingly, the present invention provides a method of assessing atrial fibrillation in a subject, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 with a reference amount, whereby atrial fibrillation can be assessed.

In one aspect of the method of the invention, the method comprises in step a) determining the amount of natriuretic peptide in a sample from the subject and in step b) comparing said amount of natriuretic peptide to a reference amount. further includes

Accordingly, the present invention provides a method of assessing atrial fibrillation in a subject, comprising:
a) determining the amount of ESM-1 and the amount of natriuretic peptides in a sample from the subject;
b) comparing said amount with a reference amount, whereby atrial fibrillation can be assessed.

An assessment of atrial fibrillation (AF) will be based on the results of the comparison step b).

Accordingly, the present invention preferably
a) determining the amount of ESM-1 and optionally natriuretic peptides in a sample from a subject;
b) comparing said amount of ESM-1 to a reference amount and optionally comparing said amount of natriuretic peptides to a reference amount;
c) assessing atrial fibrillation based on the results of said comparing step c).

The methods referred to in accordance with the present invention encompass methods consisting essentially of the above steps or methods further comprising additional steps. Furthermore, the method of the invention is preferably an ex vivo, more preferably an in vitro method. Furthermore, the method may comprise additional steps in addition to those explicitly mentioned above. For example, additional steps relate to determination of further markers and/or sample pretreatments or evaluation of the results obtained by the method. The method may be performed manually or assisted by automation. Preferably, steps (a), (b) and (c) are fully or partially automated, e.g. for measurements in step (a) or for computer-implemented calculations in step (b), using suitable robotic equipment. and sensor equipment.

According to the invention, atrial cells will be evaluated. As used herein, the term "evaluating atrial cells" refers to diagnosing atrial cells, distinguishing between paroxysmal and sustained atrial fibrillation, predicting the risk of adverse events associated with atrial fibrillation, Refers to the identification of subjects who should undergo electrocardiography (ECG) or the evaluation of treatments for atrial fibrillation.

As will be appreciated by those skilled in the art, the assessments of the present invention are generally not correct for 100% of subjects tested. The term requires that an accurate assessment (eg, diagnosis, differentiation, prognosis, identification or therapeutic assessment as referred to herein) can be performed on a statistically significant portion of subjects. Whether a portion is statistically significant can be determined by one of skill in the art without undue effort using various well-known statistical evaluation tools, such as determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc. It can be determined by Details can be found in Dowdy & Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.4, 0.1, 0.05, 0.01, 0.005 or 0.0001.

It is known that biomarkers can be elevated in various diseases and disorders. This fact also applies to ESM-1, which is known to be elevated in cancer patients, for example. However, this is considered by those skilled in the art. Thus, "assessment of atrial fibrillation" assists in the assessment of atrial fibrillation, thus in diagnosing atrial fibrillation, in distinguishing between paroxysmal and persistent atrial fibrillation. to help predict the risk of adverse events associated with atrial fibrillation, to help identify subjects who should undergo electrocardiography (ECG), or to help evaluate treatments for atrial fibrillation is interpreted as

In a preferred embodiment of the invention, the assessment of atrial fibrillation is a diagnosis of atrial fibrillation. Thus, it is diagnosed whether the subject is suffering from atrial fibrillation.

Accordingly, the present invention provides a method of diagnosing atrial fibrillation in a subject, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 with a reference amount, whereby atrial fibrillation can be diagnosed.

In one aspect, the method comprises
a) determining the amount of ESM-1 and the amount of natriuretic peptides in a sample from the subject;
b) comparing said amounts of ESM-1 and natriuretic peptides with reference amounts, whereby atrial fibrillation can be diagnosed.

Preferably, the subject tested in connection with the method of diagnosing atrial fibrillation is a subject suspected of having atrial fibrillation. However, it is also contemplated that the subject has already been diagnosed as having previously suffered from AF, and that the prior diagnosis is confirmed by practicing the methods of the invention.

In another preferred aspect of the invention, the assessment of atrial fibrillation distinguishes between paroxysmal atrial fibrillation and sustained atrial fibrillation. Thus, it is determined whether the subject is suffering from paroxysmal atrial fibrillation or persistent atrial fibrillation.

Accordingly, the present invention provides a method of distinguishing between paroxysmal and sustained atrial fibrillation in a subject, comprising:
a) measuring the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount, whereby paroxysmal atrial fibrillation and sustained atrial fibrillation can be distinguished.

In one aspect, the method comprises
a) determining the amount of ESM-1 and the amount of natriuretic peptides in a sample from the subject;
b) comparing said amount of ESM-1 and natriuretic peptide to a reference amount so that paroxysmal atrial fibrillation and sustained atrial fibrillation can be distinguished.

In another preferred aspect of the invention, the assessment of atrial fibrillation is prediction of risk of adverse events (eg, stroke) associated with atrial fibrillation. Thus, the subject is predicted to be at risk and/or not at risk of said adverse event.

Accordingly, the present invention provides a method of predicting the risk of adverse events associated with atrial fibrillation in a subject, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount, whereby the risk of adverse events associated with atrial fibrillation can be predicted.

In one aspect, the method comprises
a) determining the amount of ESM-1 and the amount of natriuretic peptides in a sample from the subject;
b) comparing said amount of ESM-1 and natriuretic peptide to a reference amount so that the risk of adverse events associated with atrial fibrillation can be predicted.

It is assumed that various adverse events can be predicted. A preferred expected adverse event is stroke.

Accordingly, the present invention is, inter alia, a method of predicting the risk of stroke in a subject, comprising:
a) measuring the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 with a reference amount, whereby the risk of stroke can be predicted.

In one aspect, the method comprises
a) determining the amount of ESM-1 and the amount of natriuretic peptides in a sample from the subject;
b) comparing said amounts of ESM-1 and natriuretic peptides with a reference amount so that the risk of stroke can be predicted.

The method may further comprise step c) of predicting stroke based on the comparison result of step b). Thus steps a), b), c) are preferably as follows:
a) measuring the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount, and
c) predicting the risk of stroke based on the comparison results of step b).

In another preferred aspect of the invention, the assessment of atrial fibrillation is assessment of a therapy for atrial fibrillation.

Accordingly, the present invention provides a method of evaluating a therapy for atrial fibrillation in a subject, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount so that a therapy for atrial fibrillation can be evaluated.

In one aspect, the method comprises
a) determining the amount of ESM-1 and natriuretic peptides in a sample from the subject;
b) comparing the amount of ESM-1 and the amount of natriuretic peptide to a reference amount so that a therapy for atrial fibrillation can be evaluated.

Preferably, the subject involved in the differentiation of atrial fibrillation, the prediction of atrial fibrillation and the evaluation of a therapy for atrial fibrillation is a subject suffering from atrial fibrillation, in particular a subject suffering from atrial fibrillation are known to have a history of atrial fibrillation (and thus are known to have a history of atrial fibrillation). However, with respect to the prediction method described above, it is also assumed that the subject has no history of atrial fibrillation.

In another preferred embodiment of the invention, the assessment of atrial fibrillation is the identification of subjects who should undergo electrocardiography (ECG). Thus, the subject is identified as who should undergo electrocardiography.

Accordingly, the present invention is a method of identifying a subject to undergo electrocardiography, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount, thereby identifying which of the subjects should undergo electrocardiography.

In one aspect, the method comprises
a) determining the amount of ESM-1 and the amount of natriuretic peptides in a sample from the subject;
b) comparing said amount of ESM-1 and natriuretic peptide to a reference amount, thereby identifying which of the subjects should undergo electrocardiography.

Preferably, the subject in the method of identifying a subject who should undergo electrocardiography is a subject with no history of atrial fibrillation. The phrase "no history of atrial fibrillation" is defined elsewhere herein.

The term "atrial fibrillation" (abbreviated AF or AFib) is well known in the art. As used herein, the term refers to supraventricular tachyarrhythmias characterized by uncoordinated atrial excitation with consequent deterioration of atrial mechanical function. In particular, the term refers to an abnormal heart rhythm characterized by frequent, irregular beats. It involves the two upper chambers of the heart. In normal heart rhythm, impulses generated by the sinoatrial node spread throughout the heart, causing contraction of the heart muscle and pumping of blood. In atrial fibrillation, the normal electrical impulses of the sinoatrial node are replaced by chaotic, rapid electrical impulses that cause an irregular heartbeat. Symptoms of atrial fibrillation are heart palpitations, fainting, shortness of breath, or chest pain. However, most episodes are asymptomatic. On the ECG, atrial fibrillation is characterized by consistent P-waves with rapid oscillations or fibrillatory excitation waves that vary in amplitude, shape and timing and are associated with irregular and frequent ventricular responses when atrial conduction is intact. Characterized by substitution.

The American College of Cardiology (ACC), the American Heart Association (AHA), and the European Society of Cardiology (ESC) have established the following classification system: (See Fuster V. et al., Circulation 2006;114: (7): e257-354; which is incorporated by reference in its entirety; see Figure 3 in the paper): primary AF, seizures persistent AF, persistent AF, and persistent AF.

All AF patients initially fall into a category called primary AF. However, the patient may or may not have previously undetected episodes. A subject has persistent AF if the AF persists for more than one year and, in particular, if a return to sinus rhythm does not occur (or occurs only upon therapeutic intervention). A subject has persistent AF if the AF persists for more than 7 days. Subjects may require either pharmacological or electrical intervention to stop atrial fibrillation. Preferably, persistent AF is symptomatic but does not spontaneously (ie, without therapeutic intervention) revert to sinus rhythm. Paroxysmal atrial fibrillation preferably refers to episodes of atrial fibrillation lasting up to 7 days. In most cases of paroxysmal AF, episodes last up to 24 hours. The development of atrial fibrillation ceases spontaneously, ie without therapeutic intervention. Thus, episodes of paroxysmal atrial fibrillation preferably terminate spontaneously, whereas episodes of persistent atrial fibrillation do not terminate spontaneously. Preferably, persistent atrial fibrillation requires electrical or pharmacological defibrillation, or other procedures, such as ablation techniques, for termination (Fuster V. et al., Circulation 2006; 114: (7): e257 -354). Both persistent and paroxysmal AF can recur, but the difference between paroxysmal and persistent AF is presented by ECG recordings: AF is considered recurrent when a patient has had two or more episodes. considered. AF, especially recurrent AF, is designated as recurrent AF if the arrhythmia spontaneously ceases. AF lasting longer than 7 days is designated as persistent.

In a preferred embodiment of the invention, the term "paroxysmal atrial fibrillation" is defined as an episode of AF that terminates spontaneously. wherein said onset lasts less than 24 hours. In another aspect, the episode that ceases spontaneously lasts up to 7 days.

A "subject" as referred to herein is preferably a mammal. Mammals include, but are not limited to, farm animals (such as cows, sheep, cats, dogs and horses), primates (including human and non-human primates such as monkeys), rabbits, and rodents (such as mice and rats). ). Preferably, the subject is a human subject.

Preferably, the tested subject is of any age, more preferably the tested subject is 50 years of age or older, more preferably 60 years of age or older, most preferably 65 years of age or older. Further, it is also envisioned that the subject being tested is 70 years of age or older.

Further, it is assumed that the tested subjects are 75 years of age or older. The subject may also be 50-90 years old.

In a preferred embodiment of the method of assessing atrial fibrillation, the subject being tested is suffering from atrial fibrillation. Thus, the subject will have a history of atrial fibrillation. Thus, the subject will have experienced an episode of atrial fibrillation prior to taking the test sample, and at least one prior episode of atrial fibrillation will have been diagnosed, eg, by ECG. For example, if the assessment of atrial fibrillation is to distinguish between paroxysmal and persistent atrial fibrillation, or if the assessment of atrial fibrillation is a prediction of the risk of adverse events associated with atrial fibrillation, or if atrial fibrillation is an evaluation of a therapy for atrial fibrillation, the patient is assumed to have atrial fibrillation.

In another preferred embodiment of the method of assessing atrial fibrillation, e.g., where the assessment of atrial fibrillation is a diagnosis of atrial fibrillation or the identification of a subject who should undergo an electrocardiogram (ECG), The subject is suspected of having atrial fibrillation.

Preferably, the subject suspected of having atrial fibrillation is a subject exhibiting at least one symptom of atrial fibrillation prior to performing the method of assessing atrial fibrillation. The symptoms are usually transient, may occur for a few seconds, and may disappear quickly. Symptoms of atrial fibrillation include dizziness, fainting, shortness of breath, and especially heart palpitations. Preferably, the subject has exhibited at least one symptom of atrial fibrillation within six months prior to taking the sample.

Alternatively or additionally, the subject suspected of having atrial fibrillation may be 70 years of age or older.

Preferably, a subject suspected of having atrial fibrillation will have no history of atrial fibrillation.

According to the present invention, a subject with no history of atrial fibrillation preferably previously, i.e. before performing the method of the present invention (especially before taking a sample from the subject), had an atrium A subject who has never been diagnosed as having fibrillation. However, the subject may or may not have an undiagnosed episode of atrial fibrillation.

Preferably, the term "atrial fibrillation" refers to all forms of atrial fibrillation. Thus, the term preferably includes paroxysmal, persistent or permanent atrial fibrillation. However, in one aspect of the invention, the tested subject does not have permanent atrial fibrillation. Thus, the term "atrial fibrillation" preferably refers only to paroxysmal and persistent atrial fibrillation.

As mentioned above, the biomarker ESM-1 can also be elevated in various diseases and disorders other than atrial fibrillation. In one aspect of the invention, it is envisaged that the subject does not have such a disease or disorder. For example, it is assumed that the subject will not have chronic kidney disease, diabetes, cancer, coronary artery disease, hypertension, and/or renal failure requiring dialysis. In one aspect, it is envisioned that the subject has no history of stroke.

In one aspect of evaluating atrial fibrillation, the subject being evaluated according to the method of evaluating atrial fibrillation does not have heart failure. The term "heart failure" is defined in relation to methods of diagnosing heart failure. Apply this definition accordingly.

In another aspect of assessing atrial fibrillation, the subject can have or is suffering from heart failure.

The subject to be tested may or may not have experienced an episode of atrial fibrillation at the time the sample was taken. Thus, in preferred embodiments of atrial fibrillation assessment (eg, diagnosis of atrial fibrillation), no episode of atrial fibrillation has been experienced at the time the sample is taken. In this aspect, the subject will have a normal sinus rhythm (and thus be in sinus rhythm) at the time the sample is taken. Thus, the diagnosis of atrial fibrillation is possible even in the (temporary) absence of atrial fibrillation. According to the method of the present invention, an elevation of endocans is preserved after the onset of atrial fibrillation, thus providing a diagnosis of a subject suffering from atrial fibrillation. Preferably, AF within about 3 days, within about 1 month, within about 3 months, or within about 6 months after performing the method of the invention (or more precisely after taking the sample). A diagnosis is made. In preferred embodiments, diagnosis of atrial fibrillation is feasible within about 6 months after onset. In preferred embodiments, diagnosis of atrial fibrillation is feasible within about 6 months after onset. Therefore, assessment of atrial fibrillation as referred to herein, in particular diagnosis, prediction or differentiation of risk as referred to herein in connection with assessment of atrial fibrillation is preferably within about 3 days, preferably within about 1 month, even more preferably within about 3 months, and most preferably within about 6 months after the last onset of movement. As a result, the sample to be tested is most preferably about 3 days after the final onset of atrial fibrillation, preferably about 1 month, more preferably about 1 month, even more preferably about 3 months. is harvested after about 6 months. Therefore, the diagnosis of atrial fibrillation preferably occurs within about 3 days, more preferably within about 3 months, and most preferably within about 6 months prior to taking the sample. contain.

However, it is assumed that the subject has experienced an episode of atrial fibrillation at the time the sample is taken (eg, for stroke prediction).

The methods of the invention can also be used to screen larger groups of subjects. Therefore, it is envisaged that at least 100, in particular at least 1000, subjects will be evaluated for atrial fibrillation. The amount of biomarker is measured in samples from at least 100, or in particular at least 1000, subjects. Further, it is envisioned that at least 10,000 subjects will be evaluated.

The term "sample" refers to a sample of bodily fluid, a sample of dissociated cells, or a sample from a tissue or organ. Samples of bodily fluids can be obtained by well-known methods and include, for example, samples of blood, plasma, serum, urine, lymph, saliva, ascitic fluid, or other humoral secretions or derivatives thereof. A tissue or organ sample can be obtained from any tissue or organ, eg, by biopsy. Separated cells can be obtained from body fluids or from tissues or organs by separation techniques such as centrifugation or cell sorting. For example, cell-, tissue- or organ samples can be taken from those cells, tissues or organs that express or produce biomarkers. Samples may be frozen, fresh, fixed (eg, formalin-fixed), centrifuged and/or embedded (eg, paraffin-embedded) samples, and the like. Cell samples, as well as various well-known post-collection preparation and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, lyophilization, ultrafiltration, concentration, etc.) may be used prior to assessing the amount of biomarkers in the sample. , evaporation, centrifugation, etc.).

In preferred embodiments of the invention, the sample is a blood (ie whole blood), serum or plasma sample. Serum is the liquid fraction of blood obtained after it has been allowed to clot. To obtain serum, the clot is removed by centrifugation and the supernatant collected. Plasma is the cell-free liquid portion of blood. To obtain plasma samples, whole blood is collected into anticoagulant-treated tubes (eg, citrated or EDTA-treated tubes). Cells are removed from the sample by centrifugation to obtain the supernatant (ie plasma sample).

As noted above, the subject may be in sinus rhythm or have an onset of AF rhythm at the time the sample is obtained.

The biomarker endothelial cell specific molecule 1 (abbreviated as ESM -1) is well known in the art. This biomarker is often also called endocan. ESM-1 is a secreted protein expressed primarily in endothelial cells of human embryonic and renal tissues. Public domain data suggest expression in heart tissue as well as in thyroid, lung and kidney. See, for example, the entry on ESM-1 in the Protein Atlas database (Uhlen M. et al., Science 2015; 347 (6220): 1260419). Expression of this gene is regulated by cytokines. ESM-1 is a proteoglycan composed of a 20 kDa mature polypeptide and 30 kDa O-linked glycan chains (Bechard D et al. J Biol Chem 2001; 276(51):48341-48349).

In a preferred embodiment of the invention, the amount of human ESM-1 polypeptide is measured in a subject's sample. The sequence of the human ESM-1 polypeptide is well known in the art (see, e.g., Lassale P. et al., J. Biol. Chem. 1996; 271:20458-20464), e.g., the Uniprot database (see entry Q9NQ30 (ESM1_HUMAN)). can be evaluated through Two isoforms of ESM-1 are produced by alternative splicing of isoform 1 (having Uniprot identifier Q9NQ30-1) and isoform 2 (having Uniprot identifier Q9NQ30-2). Isoform 1 has a length of 184 amino acids. Isoform 2 lacks amino acids 101-150 of isoform 1. Amino acids 1-19 form a signal peptide (which is cleavable).

In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, ie isoform 1 has the sequence shown under UniProt accession number Q9NQ30-1.

In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined, ie isoform 2 has the sequence shown under UniProt accession number Q9NQ30-2.

In another preferred embodiment, the amount of isoform 1 and isoform 2 of the ESM-1 polypeptide, ie the amount of total ESM-1 is determined.

For example, the amount of ESM-1 can be determined using monoclonal (eg, mouse) and/or goat polyclonal antibodies directed against amino acids 85-184 of the ESM-1 polypeptide.

The term "natriuretic peptide" encompasses atrial natriuretic peptide (ANP)-type and brain natriuretic peptide (BNP)-type peptides. Thus, natriuretic peptides according to the invention include ANP-type and BNP-type peptides and variants thereof (see, eg, Bonow RO. et al., Circulation 1996;93: 1946-1950).

ANP-type peptides include pre-proANP, proANP, NT-proANP and ANP.
BNP-type peptides include pre-proBNP, proBNP, NT-proBNP and BNP.

The pre-propeptide (134 amino acids for pre-proBNP) contains a short signal peptide that is enzymatically cleaved to release the propeptide (108 amino acids for proBNP). The propeptide is further cleaved into the N-terminal propeptide (NT-propeptide, 76 amino acids for NT-proBNP) and the active hormone (32 amino acids for BNP, 28 amino acids for ANP).

Preferred natriuretic peptides according to the invention are NT-proANP, ANP, NT-proBNP, BNP. ANP and BNP are the active hormones and have a shorter half-life than their respective inactive counterparts, NT-proANP and NT-proBNP. BNP is metabolized in the blood, whereas NT-proBNP circulates in the blood as an inactive molecule and as such is renally excreted.

The pre-analytical theory is much more robust for NT-proBNP and facilitates sample transport to central laboratories (Mueller T, Gegenhuber A, Dieplinger B, Poels W, Haltmayer M. "Long (-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in frozen plasma samples. Clin Chem Lab Med 2004; 42: 942-4).Blood samples can be stored at room temperature for several days. or can be sent or shipped without loss of recovery In contrast, storage of BNP for 48 h at room temperature or at 4° C. caused a concentration decrease of at least 20% (Mueller T, Gegenhuber A et al. Clin Chem Lab Med 2004; 42: 942-4; Wu A H, Packer M, Smith A, Bijou R, Fink D, Mair J, Wallentin L, Johnston N, Feldcamp C S, Haverstick D M, Ahnadi C E, Grant A, Despres N , Bluestein B, Ghani F. Analytical and clinical evaluation of the Bayer ADVIA Centaur automated B-type natriuretic peptide assay in patients with heart failure: a multisite study.Clin Chem 2004;50:867-73. Depending on the change or property, it may be advantageous to measure either the active or inactive form of the natriuretic peptide.

The most preferred natriuretic peptides according to the invention are NT-proBNP and BNP, especially NT-proBNP. As briefly explained above, human NT-proBNP as referred to in accordance with the present invention is preferably a polypeptide comprising 76 amino acids in length corresponding to the N-terminal portion of the human NT-proBNP molecule. The structures of human BNP and NT-proBNP have already been detailed in the prior art, e.g. WO 02/089657, WO 02/083913 and Bonow RO. et al., New Insights into the cardiac natriuretic peptides. It is described in. Preferably, the human NT-proBNP used in the present invention is human NT-proBNP as disclosed in EP 0 648 228 B1.

"Determining" the amount of a biomarker (such as ESM-1 or natriuretic peptide) as referred to herein means refers to the quantification of, for example, measuring the level of said biomarker in a sample. The terms "measuring" and "determining" are used interchangeably herein.

In one aspect, the amount of biomarker is obtained by contacting the sample with an agent that specifically binds to said biomarker, thereby forming a complex between said agent and said biomarker, and measuring the amount of the complex formed It is determined by detecting and thereby measuring the amount of said biomarker.

A biomarker (eg, ESM-1) referred to herein can be detected using methods generally known in the art. Detection methods generally include methods of quantifying the amount of biomarkers in a sample (quantification methods). A person skilled in the art will generally know which of the following methods are suitable for the qualitative and/or quantitative detection of biomarkers. Samples can be conveniently assayed for proteins, eg, using Western methods and immunoassays, such as ELISA, RIA, fluorescence and luminescence-based immunoassays and proximity expansion assays, which are commercially available. Further suitable methods for detecting biomarkers include measuring physical or chemical properties specific to the peptide or polypeptide, such as its precise molecular mass or NMR spectrum. The methods include analytical devices such as, for example, biosensors, optical devices coupled to immunoassays, biochips, mass spectrometers, NMR analyzers or chromatographic devices. In addition, the methods include microplate ELISA, fully automated or robotic immunoassays (e.g. available on Elecsys ^™ analyzer), CBA (enzymatic cobalt binding assay Cobalt Binding Assay; e.g. on Roche-Hitachi ^™ analyzer). available) and latex agglutination assays (eg available on the Roche-Hitachi ^™ analyzer).

A wide variety of immunoassay techniques, using assay formats such as those described above, are available for the detection of the biomarker proteins referred to herein. See, for example, US Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include conventional competitive binding assays as well as non-competitive one- and two-site or "sandwich" assays. These assays also include direct binding of labeled antibodies to target biomarkers.

Methods of using electrochemiluminescent labels are also well known. Such methods exploit the ability of special metal complexes to decay from an excited state to a ground state upon oxidation and emit electrochemiluminescence. For a review see Richter, M.M., Chem. Rev. 2004; 104: 3003-3036.

In one aspect, a detection antibody (or an antigen-binding fragment thereof) that can be used to measure the amount of a biomarker is ruthenium or iridium. Accordingly, the antibody (or antigen-binding fragment thereof) will contain a ruthenium label. In one aspect, the ruthenium label is a bipyridine-ruthenium(II) complex. Alternatively, the antibody (or antigen-binding fragment thereof) will contain an iridium label. In one embodiment, said iridium label is a complex as disclosed in WO 2012/107419.

In one embodiment of a sandwich assay for measuring ESM-1, the assay comprises a biotinylated primary monoclonal antibody (as the capture antibody) that specifically binds ESM-1 and a secondary monoclonal that specifically binds ESM-1. and a ruthenium F(ab')2 fragment of the antibody (as detection antibody). The two antibodies form a sandwich-type immunoassay complex with ESM-1 in the sample.

In one embodiment of a sandwich assay for the determination of natriuretic peptides, the assay comprises a biotinylated primary monoclonal antibody (as a capture antibody) that specifically binds natriuretic peptides, and a secondary monoclonal antibody that specifically binds natriuretic peptides. Ruthenium F(ab')2 fragments of the following monoclonal antibodies (as detection antibodies). The two antibodies form a sandwich-type immunoassay complex with natriuretic peptides in the sample.

Determining the amount of a polypeptide (such as ESM-1 or a natriuretic peptide) preferably comprises the steps of (a) contacting said polypeptide with an agent that specifically binds said polypeptide, (b) (optionally b) removing unbound agent; and (c) measuring the amount of bound binding agent, ie the amount of complexes of agents formed in step (a). According to a preferred embodiment, said contacting, removing and measuring steps may be preformed by an analyzer device. According to an aspect, the steps may be performed by a single analyzer unit of the system or by multiple analyzer units operatively communicatively connected to each other. For example, in certain embodiments, the systems disclosed herein include a first analyzer unit for performing the contacting and removing steps, and a transport unit (e.g., a robotic arm) to the first analyzer unit. and a second analyzer unit operatively connected in communication.

An agent that specifically binds a biomarker (also referred to herein as a "binding agent") is covalently or non-covalently coupled to a label that allows detection and measurement of the bound agent. may be ringed. Labeling can be done by direct or indirect methods. Direct labeling involves coupling a label directly (covalently or non-covalently) to a first binding agent. Indirect labeling involves binding (covalently or non-covalently) a second binding agent to the first binding agent. The second binding agent should bind specifically to the first binding agent. Said second binding agent may be linked to a suitable label and/or be the target (receptor) of a third binding agent that binds to the second binding agent. Suitable second and higher order binding agents include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). A binding agent or substrate can also be "tagged" with one or more tags as known in the art. Such tags may be targets for higher order binding agents. Suitable tags include biotin, digoxigenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein and the like. For peptides or polypeptides, the tag is preferably at the N-terminus and/or C-terminus. A suitable label is any label detectable by a suitable detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium complexes, iridium complexes, enzymatically active labels, radioactive labels, magnetic labels ("magnetic beads" such as paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and derivatives thereof. Suitable detection substrates include diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-yne drill phosphate; available as ready-made stock solutions from Roche Diagnostics), CDP-Star ^™ (Amersham Biosciences), ECF ^™ (Amersham Biosciences). Suitable enzyme-substrate combinations may result in colored reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art (eg, using light-sensitive film or a suitable imaging system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (eg GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and Alexa dyes (eg Alexa 568). Additional fluorescent labels are available, for example, from Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also envisioned. Radiolabels can be detected by any method known and suitable, such as light-sensitive film or fluorophotography.

The amount of polypeptide may also preferably be determined as follows: (a) a solid support comprising a binding agent for the polypeptide as described herein, containing the peptide or polypeptide; contacting with a sample; and (b) measuring the amount of peptide or polypeptide bound to said support. Materials for manufacturing supports are well known in the art, in particular column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips, as well as surfaces, nitrocellulose strips, membranes. , sheets, duracites, wells and walls of reaction trays, plastic tubes, and the like.

In another aspect, the sample is removed from complexes formed between the binding agent and the at least one marker prior to measuring the amount of complexes formed. Thus, in one aspect the binding agent may be immobilized on a solid support. In yet another aspect, the sample can be removed from complexes formed on the solid support by applying a wash solution.

"Sandwich assays" are among the most useful and commonly used assays and include many variations of the sandwich assay technique. Briefly, in a typical assay, an unlabeled (capture) binding agent is or can be immobilized on a solid support, and the sample to be tested is contacted with said capture binding agent. After a suitable incubation period for a period of time sufficient to allow formation of binding agent-biomarker complexes, a second (detection) binding labeled with a reporter molecule capable of producing a detectable signal. The agent is added and incubated, allowing sufficient time for the formation of another binding agent-biomarker-labeled binding agent complex. The presence of biomarkers is determined by washing away any unreacted material and observing the signal produced by the reporter molecule bound to the detection binding agent. Results may be qualitative by simple observation of the visible signal or quantitative by comparison to control samples containing known amounts of biomarkers.

The incubation steps of a typical sandwich assay can be varied as necessary and appropriate. Such variations include, for example, co-incubation of two or more binding agents and biomarkers together. For example, the sample to be analyzed and the labeled binding agent are added simultaneously to the immobilized capture binding agent. It is also possible to first incubate the sample to be analyzed and the labeled binding agent before adding the antibody bound to the solid phase or capable of binding to the solid phase.

The complex formed between the specific binding agent and biomarker should be proportional to the amount of biomarker present in the sample. The specificity and/or sensitivity of the applied binding agent limits the extent to which the proportion of at least one marker contained in the sample can specifically bind. Details on how the measurements can be performed can be found in this specification. The amount of complex formed will be converted to an amount of biomarker that reflects the amount actually present in the sample.

The terms "binding agent", "specific binding agent", "analyte-specific binding agent", "detection agent" and "agent that specifically binds to a biomarker" are used interchangeably. Preferably, the term relates to agents comprising binding moieties that specifically bind the corresponding biomarkers. Examples of "binding agents", "detecting agents", "agents" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs) or chemical compounds. A preferred agent is an antibody that specifically binds to the biomarker to be measured. The term "antibody" is used in the broadest sense and encompasses various antibody structures such as, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and desired antibody fragments (ie, antigen-binding fragments derived therefrom) are included as long as they exhibit the antigen-binding activity of Preferably, the antibody is a polyclonal antibody (or antigen-binding fragment thereof). More preferably, the antibody is a monoclonal antibody (or antigen-binding fragment thereof). It is also envisioned that two monoclonal antibodies that bind to different locations on ESM-1 are used (in a sandwich assay), as described herein. Thus, at least one antibody is used to determine ESM-1 levels.

In one aspect, at least one antibody is a murine monoclonal antibody. In another aspect, at least one antibody is a rabbit monoclonal antibody. In yet another aspect, the antibody is a goat polyclonal antibody. In yet another aspect, the antibody is a sheep polyclonal antibody.

The terms "specific binding" or "binds specifically" refer to binding reactions in which binding pair molecules exhibit binding to each other under conditions that do not significantly bind to other molecules. The term “specific binding” or “binds specifically” when referring to proteins or peptides as biomarkers preferably binds the binding agent with an affinity (“association constant” K _a ) of at least 10 ⁷ M ⁻¹ . Refers to the binding reaction when binding to the corresponding biomarker. The term "specific binding" or "binds specifically" preferably refers to an affinity of at least ¹⁰⁸ M ^{-1 ,} more preferably of at least ¹⁰⁹ M-1 for its target molecule. The terms "specifically" or "specifically" are used to indicate that other molecules present in the sample do not significantly bind to the binding agent specific for the target molecule.

In one aspect, the methods of the invention are based on detecting protein complexes comprising human ESM-1 and non-human or chimeric ESM-1 specific binding agents. In such embodiments, the invention provides a method of assessing atrial fibrillation in a subject, said method comprising the step of (a) incubating a sample from said subject with a non-human ESM-1 specific binding agent. (b) measuring the complex between the ESM-1 specific binding agent and ESM-1 formed in (a); and (c) comparing said measured amount of complex to a reference amount. including. An amount of conjugate equal to or greater than the reference amount is indicative of the diagnosis (and thus existence) of atrial fibrillation and of subjects who should have an ECG or who are at risk of adverse events. An amount of conjugate below the reference amount is indicative of the absence of atrial fibrillation, the presence of paroxysmal atrial fibrillation, subjects who may not undergo an ECG, or subjects who are not at risk of adverse events.

The term "amount" as used herein refers to the absolute amount of a biomarker (eg ESM-1 or natriuretic peptide) as referred to herein, the relative amount or concentration of said biomarker as well as It includes any value or parameter that can be correlated or derived therefrom. Such values or parameters include intensity signal values of all specific physical or chemical properties obtained from said peptides by direct measurements, eg intensity values in mass spectra or NMR spectra. In addition, all values or parameters obtained by indirect measurements detailed herein, e.g., the amount of response measured from a biological readout (output) device in response to the peptide, or Also included is the intensity signal obtained from the ligand. It should be understood that any standard mathematical operation can also yield values correlating to the quantities or parameters mentioned above.

As used herein, the term "compare" refers to the amount of a biomarker (e.g., ESM-1 and a natriuretic peptide, e.g., NT-proBNP or BNP) in a sample of a subject identified herein. It refers to comparing to a reference amount of a biomarker that has been identified. Comparison, as used herein, refers to the comparison of corresponding parameters or values, e.g., absolute amounts are compared to absolute reference amounts, while concentrations are compared to reference concentrations, or intensity signals obtained from biomarkers in samples. is compared with the same kind of intensity signal obtained from a reference sample. Comparison can be manual or computer-assisted. The comparison can thus be performed by a computer device. A numerical value of a measured or detected amount of a biomarker in a sample of a subject and a numerical value of a reference amount, for example, can be compared to each other, and said comparison can be automatically performed by a computer program that performs the operations for comparison. can be implemented. A computer program that implements the evaluation will provide the desired evaluation in a suitable output format. In the case of computer-assisted comparisons, the values of the measured quantities are compared by a computer program to values corresponding to suitable references stored in a database. The computer program can further evaluate the results of the comparison, ie automatically provide the desired evaluation in a suitable output format. In the case of computer-assisted evaluation, the values of the measured quantities are compared by a computer program with values corresponding to suitable references stored in a database. The computer program can further evaluate the results of the comparison, ie automatically provide the desired evaluation in a suitable output format.

According to the invention, the biomarker ESM-1 amount and optionally the natriuretic peptide amount are compared with a reference. The reference is preferably a reference amount. The term "reference amount" is well understood by those skilled in the art. It should be understood that the reference amount is provided to enable the assessment of atrial fibrillation as described herein. For example, in the context of a method of diagnosing atrial fibrillation, the reference amount is preferably (i) a group of subjects suffering from atrial fibrillation, or (ii) a group of subjects not suffering from atrial fibrillation. refers to the amount that allows assignment of a subject to either A suitable reference amount can be determined from a reference sample to be analyzed together with the test sample, ie simultaneously or sequentially.

It should be understood that the amount of ESM-1 is compared to a reference amount of ESM-1, while the amount of natriuretic peptide is compared to a reference amount of natriuretic peptide. If the amounts of two markers are determined, it is also envisioned to calculate a combined score based on the amounts of ESM-1 and natriuretic peptides. In a subsequent step the score is compared to a reference score.

A reference amount can theoretically be calculated for the cohort of subjects described above based on the mean value of a given biomarker by applying standard statistical methods. In particular, the accuracy of a test, such as a method aimed at diagnosing the presence or absence of an event, is best described by its Receiver Operating Characteristic (ROC) (particularly Zweig MH. et al., Clin. Chem. 1993; 39: 561-577). A ROC graph is a plot of all sensitivity versus specificity pairs obtained from continuously varying the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, ie the ability to correctly assign subjects to a given prognosis or diagnostic method. The ROC plot shows the overlap between the two distributions by plotting sensitivity against 1-(minus) specificity for the full range of thresholds suitable for making the distinction. The y-axis is the sensitivity, or true positive rate, defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. The x-axis is the false positive rate, or 1-(minus) specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative results and the number of false positive results. It is a measure of specificity and is calculated from the completely unaffected subgroup. The ROC plot is independent of the prevalence of events in the cohort because the true positive and false positive rates are calculated completely separately using test results from two different subgroups. Each point on the ROC plot represents a sensitivity/1-(minus) specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two outcome distributions) is the top leftmost, which has a true positive rate of 1.0 or 100% (perfect sensitivity) and a false positive rate of 0 (perfect specificity). It has an ROC plot that goes through a point, which is in degrees). The threshold plot for a test with no discrimination (results in the two groups have identical distributions) is a 45° diagonal line starting from the lower left edge and extending to the upper right edge. Most plots fall between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily corrected by reversing the "positive" criteria from "greater than" to "less than" (or vice versa). be. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Depending on the confidence interval desired, threshold values can be derived from the ROC curve using the appropriate balance of sensitivity and specificity, respectively, to allow diagnosis for a given event. Therefore, preferably, an ROC curve is generated for said cohort as described above, and a threshold dose is derived therefrom, to allow a reference that can be used in the methods of the invention, namely the assessment of atrial fibrillation. You can create a threshold that For example, optimal sensitivity is desired for excluding (i.e., excluding) subjects with atrial fibrillation, while optimal specificity is for subjects assessed as having atrial fibrillation. is assumed to be In one aspect, the methods of the invention allow prediction of whether a subject is at risk for atrial fibrillation and/or adverse events associated with atrial fibrillation, such as the development or recurrence of stroke.

In preferred embodiments, the term "reference amount" refers to a predetermined value. Said predetermined value allows to assess atrial fibrillation, thus diagnosing atrial fibrillation, distinguishing between paroxysmal and sustained atrial fibrillation, and reducing the risk of adverse events associated with atrial fibrillation. It would predict risk, identify subjects who should undergo electrocardiography (ECG), or allow evaluation of treatments for atrial fibrillation. It should be understood that the reference amount may vary based on the type of evaluation. For example, the reference amount of ESM-1 for discrimination of AF will generally be higher than the reference amount for diagnosis of AF. However, this will be taken into consideration by those skilled in the art.

As noted above, the term "evaluating atrial fibrillation" preferably includes the diagnosis of atrial fibrillation, the distinction between paroxysmal and persistent atrial fibrillation, and the risk of adverse events associated with atrial fibrillation. Refers to prediction, identification of patients who should have an electrocardiogram (ECG), or evaluation of treatment for atrial fibrillation. In the following, these embodiments of the method of the invention are described in more detail. The above definitions apply accordingly.

Methods of Diagnosing Atrial Fibrillation The term "diagnosing" as used herein refers to assessing whether a subject is suffering from atrial fibrillation (AF) when referred to according to the methods of the present invention. means. In one aspect, the subject is diagnosed as having AF. In preferred embodiments, the subject is diagnosed as having paroxysmal AF. In another embodiment, the subject is diagnosed as not having AF.

All types of AF can be diagnosed according to the present invention. Atrial fibrillation can be paroxysmal, persistent or permanent AF. Preferably, paroxysmal or atrial fibrillation is diagnosed in a subject not particularly suffering from persistent AF.

The actual diagnosis of whether a subject has AF may involve additional steps such as confirmation of the diagnosis (eg, by ECG such as Holter-ECG). The present invention allows assessment of the likelihood that a patient is suffering from atrial fibrillation. Subjects with ESM-1 amounts above the reference amount are likely to have atrial fibrillation, while subjects with ESM-1 amounts below the reference amount are likely to have atrial fibrillation. do not have. Thus, the term "diagnosing" in the context of the present invention also includes assisting a physician in assessing whether a subject is suffering from atrial fibrillation.

Preferably, an increased amount of ESM-1 (and optionally natriuretic peptide amount) in a sample from a test subject compared to a reference amount(s) is indicative of a subject suffering from atrial fibrillation. and/or an amount of ESM-1 (and optionally an amount of natriuretic peptide) in a sample from a test subject that is decreased compared to the reference amount(s) compared to subjects not suffering from atrial fibrillation It is an index of the body.

In a preferred embodiment, the reference amount, ie the ESM-1 reference amount and, if natriuretic peptides are measured, the reference amount of natriuretic peptides are in subjects suffering from atrial fibrillation and It will make it possible to distinguish between subjects who are not Preferably, said reference amount is a defined value.

In one aspect, the methods of the invention allow diagnosis of a subject suffering from atrial fibrillation. Preferably, if the ESM-1 amount (and optionally the natriuretic peptide amount) is above the reference amount, the subject is probably suffering from atrial fibrillation. In one aspect, the subject has atrial fibrillation if the ESM-1 amount is above the upper reference limit (URL) of some percentile (e.g., the 99th percentile) of the reference amount. do not have.

In another preferred embodiment, the method of the invention allows for the diagnosis that a subject is not suffering from atrial fibrillation. Preferably, if the ESM-1 amount (and optionally the natriuretic peptide amount) is below the reference amount (a certain percentile URL), the subject does not have AF. Thus, in one aspect, the term "diagnose atrial fibrillation" refers to "rule out atrial fibrillation."

Excluding atrial fibrillation is of particular interest because it avoids additional diagnostic tests for diagnosing atrial fibrillation, such as ECG examinations. Thus, the present invention avoids unnecessary medical costs.

Accordingly, the present invention is a method of ruling out atrial fibrillation comprising:
a) determining the amount of ESM-1 in the subject's sample, and
b) comparing said amount of ESM-1 with a reference amount, whereby atrial fibrillation is ruled out.

Preferably, a decreased amount of the biomarker ESM-1 in the subject's sample compared to a reference amount (e.g., a reference to rule out atrial fibrillation) is indicative of a subject not suffering from atrial fibrillation. and thus an indicator to rule out atrial fibrillation in that subject. For example, a reference amount of ESM-1 can be determined in a sample of subjects not suffering from AF, or from a sample of a group of subjects thereof.

A combined determination of the biomarkers ESM-1 and natriuretic peptides can achieve an even more reliable exclusion. Therefore steps a) and b) are preferably as follows:
a) determining the amount of ESM-1 and natriuretic peptides in a sample from the subject, and
b) comparing the amounts of ESM-1 and natriuretic peptides with reference amounts, thereby ruling out atrial fibrillation.

Preferably, the amounts of the two biomarkers, namely the biomarker ESM-1 amount and natriuresis, are reduced in the subject's sample compared to their respective reference amounts (e.g., the reference amount to rule out atrial fibrillation). The amount of peptide is an indicator of a subject not suffering from atrial fibrillation and thus an indicator of ruling out atrial fibrillation in the subject. For example, a reference amount of a natriuretic peptide can be determined in a sample of subjects not suffering from AF, or from a sample of a group of subjects thereof.

In one aspect of the method of diagnosing atrial fibrillation, the method further comprises recommending and/or initiating a therapy for atrial fibrillation based on the diagnostic results. Preferably, treatment is recommended or initiated when a subject is diagnosed as having AF. A preferred method of treating atrial fibrillation is disclosed herein.

Methods of Distinguishing Between Paroxysmal and Persistent Atrial Fibrillation The term "distinguish" as used herein means distinguishing between paroxysmal and persistent atrial fibrillation in a subject. . The term as used herein preferably includes differential diagnosis of paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject. Thus, the methods of the present invention allow a subject with atrial fibrillation to assess whether they are suffering from paroxysmal atrial fibrillation or persistent atrial fibrillation. The actual distinction may involve additional steps such as confirmation of the distinction. Therefore, the term "distinguish" according to the present invention also includes helping physicians distinguish between paroxysmal AF and persistent AF.

Preferably, an increased amount of ESM-1 (and optionally the amount of natriuretic peptide) in a sample from a subject compared to the reference amount (or amounts) is associated with persistent atrial fibrillation. the amount of ESM-1 (and optionally the amount of natriuretic peptide) in a sample from a subject that is indicative of a subject who is living and/or is decreased compared to a reference amount (or amounts) is indicative of subjects not suffering from paroxysmal atrial fibrillation. In both AF types (paroxysmal and persistent), ESM-1 levels are increased compared to reference levels in non-AF subjects.

In preferred embodiments, the reference amount will allow discrimination between subjects suffering from paroxysmal atrial fibrillation and subjects suffering from persistent atrial fibrillation. Preferably said reference amount is a defined amount.

In one aspect of the method of distinguishing between paroxysmal and persistent atrial fibrillation, the subject does not have persistent atrial fibrillation.

Methods of Predicting the Risk of Adverse Events Associated with Atrial Fibrillation The methods of the present invention also relate to methods of predicting the risk of adverse events.
In one aspect, the risk of adverse events as described herein can be a prediction of any adverse event associated with atrial fibrillation. Preferably, said adverse event is selected from recurrence of atrial fibrillation (eg recurrence after cardioversion) and stroke. Therefore, the risk of a subject (atrial fibrillation sufferer) suffering from adverse events (such as stroke or recurrence of atrial fibrillation) in the future would be predicted.

Further, it is expected that said adverse event associated with atrial fibrillation is the development of atrial fibrillation in a subject with no history of atrial fibrillation.

In particularly preferred embodiments, the risk of stroke is predicted.
Accordingly, the present invention provides a method of predicting stroke risk in a subject, comprising:
a) determining the amount of ESM-1 in the subject's sample, and
b) comparing said amount of ESM-1 with a reference amount, whereby the risk of stroke can be predicted.

Preferably, the term "predict risk" as used herein refers to the likelihood (probability) that a subject will suffer an adverse event (e.g., stroke) as referred to herein. about evaluating Typically, it is predicted whether a subject is at risk (high risk) or not at risk (low risk) of said adverse event. Thus, the method of the invention makes it possible to distinguish between subjects at risk of suffering from said adverse event and subjects who are not at risk. The method of the invention allows to distinguish between subjects who are low risk, average risk or high risk.

As mentioned above, the risk (and likelihood) of suffering said adverse event will be predicted in a certain time window. In preferred aspects of the invention, the prediction window is about 3 months, about 6 months, or about 1 year. In another preferred embodiment, the prediction window is a period of about 5 years (eg, for stroke prediction). Further, the prediction window may be a period of about 6 years (eg, for stroke prediction). Alternatively, the prediction window may be a period of approximately 10 years. It is also envisioned that the prediction window is for a period of 1 to 10 years.

Preferably, said prediction window is calculated from the completion of the method of the invention. More preferably, said prediction window is calculated from the time of acquisition of the sample to be tested. As will be appreciated by those skilled in the art, risk predictions are generally not intended to be correct in 100% of subjects. However, the term requires that predictions can be made for a statistically significant portion of subjects in an appropriate and correct manner. Whether a given fraction is statistically significant can be determined without further elaboration by those skilled in the art using various well-known statistical evaluation methods such as determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney (Mann-Whitney) test or the like can be used for determination. Details can be found in Dowdy & Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005 or 0.0001.

In a preferred embodiment, the phrase "predicts the risk of suffering said adverse event" means that the subject to be analyzed by the method of the invention is at risk of suffering said adverse event (e.g. stroke). Means to be assigned either to a group or to a group of subjects who are not at risk of suffering said adverse event. Thus, it is predicted whether a subject is at risk or not at risk of suffering said adverse event. A "subject at risk of suffering said adverse event" as used herein is preferably at high risk (preferably within the predictive window) of suffering said adverse event. Preferably, said risk is high compared to the average risk in the cohort of subjects. A "subject not at risk of suffering said adverse event" as used herein preferably has a low risk (preferably within the predictive window) of suffering said adverse event. Preferably, said risk is low compared to the average risk in a cohort of subjects. Subjects at risk of suffering said adverse event preferably have at least a 20% risk of suffering said adverse event, such as recurrence or development of atrial fibrillation, preferably within a predictive window of about 1 year, or more preferably At least 30%. Subjects who are not at risk of suffering said adverse event preferably have a risk of suffering said adverse event of less than 12%, more preferably less than 10%, within a predictive window of 1 year.

With respect to predicting stroke, a subject at risk of suffering said adverse event preferably has a risk of suffering said adverse event of at least 10% within a predictive window of about 5 years, particularly within a predictive window of about 6 years. or more preferably at least 13%. Subjects who are not at risk of suffering said adverse event preferably have a risk of suffering said adverse event of less than 10%, more preferably 8 within a predictive window of about 5 years, especially within a predictive window of about 6 years. %, most preferably less than 5%. The risk will be even higher if the subject does not receive anticoagulant therapy. This will be taken into consideration by those skilled in the art.

Preferably, an increased amount of ESM-1 (and optionally the amount of natriuretic peptide) in a sample from a subject compared to the reference amount(s) is associated with risk of adverse events associated with atrial fibrillation. an amount of ESM-1 (and optionally an amount of natriuretic peptide) in a sample from a subject that is indicative of a subject and/or decreased compared to a reference amount(s) is determined in the atrium It is an indicator of subjects at no risk of adverse events associated with fibrillation.

In preferred embodiments, the reference amount will allow distinguishing between subjects at risk of an adverse event as referred to herein and subjects not at risk of said adverse event. Preferably said reference amount is a predetermined value.

In preferred embodiments of the above methods, the risk of stroke is predicted. Preferably said stroke will be associated with atrial fibrillation. More preferably the stroke is induced by atrial fibrillation.

Preferably, stroke is associated with atrial fibrillation if there is a temporal relationship between one episode of stroke and atrial fibrillation. More preferably, if the stroke can be induced by atrial fibrillation, the stroke is associated with atrial fibrillation. For example, atrial fibrillation can lead to cardioembolic stroke (often also referred to as embolic or thrombotic stroke). Preferably, oral anticoagulant therapy can prevent stroke associated with AF.
A predictable stroke is preferably a cardioembolic stroke.

Also preferably, the stroke is considered to be associated with atrial fibrillation if the subject to be tested has and/or has a history of atrial fibrillation. Also, in one aspect, if a subject is suspected of having atrial fibrillation, the stroke may be considered associated with atrial fibrillation.

The terms "compare," "amount," "subject," and "determine," etc. are defined herein. These definitions apply accordingly. For example the sample is preferably a blood, serum or plasma sample.

In a preferred embodiment of said method of predicting an adverse event (eg stroke), the subject to be tested has atrial fibrillation. More preferably, the subject has a history of atrial fibrillation. According to the method of predicting an adverse event, the subject preferably has persistent atrial fibrillation, more preferably persistent atrial fibrillation, and most preferably paroxysmal atrial fibrillation.

In one aspect of the method of predicting an adverse event, a subject with atrial fibrillation has experienced an episode of atrial fibrillation at the time the sample is taken. In another aspect of the method of predicting an adverse event, the patient with atrial fibrillation has not experienced an episode of atrial fibrillation (and thus has a normal sinus rhythm) at the time the sample is taken. Additionally, the subject whose risk is to be predicted may be on anticoagulant therapy.

In another embodiment of the method of predicting adverse events, the subject to be tested has no history of atrial fibrillation. In particular it is assumed that the subject does not suffer from atrial fibrillation.

Studies supporting the present invention further demonstrated that measurement of ESM-1 can improve the accuracy of predicting a subject's clinical stroke risk score. Thus, combined measures of clinical stroke risk score and ESM-1 measure allow significantly more reliable prediction of stroke compared to either ESM-1 measure alone or clinical stroke risk score measure alone (implemented See Example 6).

Accordingly, methods of predicting stroke risk further include combining ESM-1 levels with clinical stroke risk scores. A test subject's risk of developing stroke is predicted based on the combination of ESM-1 amount and clinical risk score.

In one aspect of the above method, the method further comprises comparing the amount of ESM-1 to a reference amount. In this case, the results of the comparison are combined with a clinical stroke risk score.

Accordingly, the present invention provides, inter alia, a method for predicting the risk of developing a stroke in a subject, comprising:
a) determining the amount of ESM-1 in the subject's sample, and
b) combining said ESM-1 amount with a clinical stroke risk score so that said subject's stroke risk can be predicted.

According to this method, the subject is assumed to be one with a known clinical stroke risk score. Therefore, the clinical stroke risk score value is known for the subject.

Accordingly, the method may comprise obtaining or providing a clinical stroke risk score value. Preferably the value is a number. In one aspect, a clinical stroke risk score is generated by one of the clinical-based tools commonly available to physicians. Preferably, said value is provided by determining a clinical stroke risk score value for each subject. More preferably, the value is obtained from a patient record database or the subject's medical history. Therefore, the value of the score can also be determined using subject's historical data or published data.

According to the present invention, ESM-1 quantity is combined with a clinical stroke risk score. This means that the ESM-1 amount value is preferably combined with a clinical stroke risk score. Accordingly, those values are operatively combined to predict a subject's risk of suffering a stroke. By combining values, a single value can be calculated, which itself can be used for prediction.

Clinical stroke risk scores are well known in the art. For example, the scores are described in Kirchhof P. et al. (European Heart Journal 2016; 37: 2893-2962), which is incorporated herein by reference for its entire disclosure. In one aspect, the score is the CHA2DS2 _{- VASc} score. In another embodiment, the score is a CHADS ₂ score (Gage BF. et al., JAMA, 285 (22) (2001), pp. 2864-2870) and an ABC score (Hijazi Z. et al., Lancet 2016; 387 (10035): 2302 -2311).

Methods for Improving Accuracy of Predicting Clinical Stroke Risk Score The invention further provides a method of improving the accuracy of predicting a clinical stroke risk score for a subject, comprising:
a) determining the amount of ESM-1 in the sample, and
b) combining said ESM-1 amount with a clinical stroke risk score, thereby improving the predictive accuracy of said clinical stroke risk score.

The method may further comprise the step of c) improving the predictive accuracy of said clinical stroke risk score based on the results of step b).

The definitions and explanations given above in relation to methods of assessing atrial fibrillation, in particular of predicting the risk of adverse events (eg stroke), preferably apply to the above methods as well. For example, the subject is assumed to be one with a known clinical stroke risk score. Alternatively, the method may comprise obtaining or providing a clinical stroke risk score value.

According to the present invention, ESM-1 quantity is combined with a clinical stroke risk score. This means that the ESM-1 amount value is preferably combined with the clinical stroke risk score. Accordingly, those values are operatively combined to improve the predictive accuracy of said clinical stroke risk score.

Methods of Identifying Patients Who Should Have Electrocardiography (ECG) According to this aspect of the method of the invention, a subject to be tested with a biomarker undergoes electrocardiography (ECG), an electrocardiogram evaluation. should be evaluated. Said evaluation will be performed diagnostically, ie to detect the presence or absence of AF, in said subject.

As used herein, the term "identifying a subject" utilizes information or data generated relating to the amount of ESM-1 in a sample of the subject to identify a patient who should undergo an ECG. point to Subjects identified have a high likelihood of having AF. An ECG evaluation is performed as confirmation.

Electrocardiography (abbreviated ECG) is a method of recording the electrical activity of the heart by means of a suitable ECG. An ECG machine records electrical signals produced by the heart that extend throughout the body, down to the skin. Recording of electrical signals is accomplished by connecting electrodes contained in an ECG device to the subject's skin. The method of obtaining the records is non-invasive and risk-free. An ECG is performed to make a diagnosis of atrial fibrillation, eg, to assess the presence or absence of atrial fibrillation in a test subject. In one aspect of the methods of the present invention, the ECG device is a one-lead device (eg, a one-lead handheld (manually operated) ECG device). In another preferred embodiment, the ECG device is a 12-lead ECG device, such as a Holter monitor.

Preferably, the amount of ESM-1 in the test subject's sample increased compared to the reference amount(s) is indicative of the subject to undergo an ECG and/or the reference amount ( A decreased amount of ESM-1 in a subject's sample compared to one or more) is indicative of a subject who may not undergo an ECG.

In preferred embodiments, the reference amount will allow distinguishing between subjects who should have an ECG and those who should not. Preferably, said reference amount is a predetermined amount.

Methods of Evaluating Therapy for Atrial Fibrillation As used herein, the term "evaluating therapy for atrial fibrillation" preferably refers to the evaluation of therapy aimed at treating atrial fibrillation. .

The therapy being evaluated can be any therapy aimed at treating atrial fibrillation. Preferably, said therapy is selected from the group consisting of at least one anticoagulant, sinus rhythm maintenance (rhythm control), heart rate regulation (rate control), defibrillation and ablation. Such therapies are well known in the art and are reviewed, for example, in Fuster V et al., Circulation 2011; 123: e269-e367, the entire contents of which are incorporated herein by reference.

In one aspect, the therapy is administration of at least one anticoagulant. Administration of at least one anticoagulant is aimed at reducing or preventing blood clotting and associated stroke. In one aspect, the at least one anticoagulant is heparin, coumarin derivatives such as warfarin or dicoumarol, tissue factor pathway blockers (TFPI), antithrombin III, factor IXa inhibitors, factor Xa inhibitors, factor Va and Inhibitors of factor VIIIa, as well as thrombin inhibitors (anti-type IIa).

In a preferred embodiment, evaluating therapy for atrial fibrillation is monitoring said therapy. In this embodiment, the reference amount is preferably the amount of EMS-1 in an early sample taken (ie a sample taken earlier than the test sample in step a).
Optionally, the amount of natriuretic peptide is determined in addition to the amount of ESM-1.

Accordingly, the present invention provides a method of monitoring therapy for atrial fibrillation in a subject, said subject preferably suffering from atrial fibrillation, said method comprising
a) determining the amount of ESM-1 (and optionally the amount of natriuretic peptides) in a sample from said subject; and
b) comparing said amount of ESM-1 to a reference amount, wherein said reference amount is the amount of ESM-1 in a sample taken from said subject prior to said sample in step a), and optionally comparing the amount of natriuretic peptide to a reference amount, wherein said reference amount is the amount of natriuretic peptide in a sample obtained from said subject prior to said sample in step a). on how to.

The sample in step a) is referred to herein as the "test sample" and the sample in step b) is referred to herein as the "reference sample".

The term "monitoring" preferably relates to assessing the efficacy of therapy as referred to herein. Thus, the efficacy of therapy (eg, anticoagulant therapy) is monitored.

The above method may comprise the additional step of monitoring therapy based on the results of the comparison step, performed in step c). As will be appreciated by those skilled in the art, risk predictions are generally not correct for 100% of subjects. However, the term requires that the prediction can be performed on a statistically significant proportion of subjects in an appropriate and correct manner. Therefore, the actual monitoring may further include additional steps such as confirmation.

Preferably, by practicing the methods of the invention, it is possible to assess whether a subject will respond to said therapy. If the subject's condition improves between taking the first and second samples, the subject responds to the therapy. Preferably, if the condition worsens between taking the first and second samples, the subject does not respond to the therapy.

Preferably, a reference sample is obtained prior to initiation of said therapy. More preferably, the sample is taken within 2 weeks, especially within 1 week, prior to initiation of said therapy. However, it is also envisioned that the reference sample is obtained after the therapy has been initiated (but before the test sample is obtained). In this case, ongoing therapy is monitored.

Therefore, the test sample should be obtained after the reference sample. It should be understood that the test sample is obtained after initiation of said therapy.

Additionally, the test sample is taken after a reasonable amount of time after the reference sample is taken. It should be understood that the amounts of biomarkers described herein do not change rapidly (eg, within 1 minute or 1 hour). Therefore, "moderate" in this case refers to the interval between taking the first sample and the test sample such that the biomarkers can be adjusted. Thus, the test sample is preferably obtained at least 1 month, at least 3 months or especially at least 6 months after said reference sample.

Preferably, an increase, more preferably a significant increase, most preferably an increase in the amount of biomarkers in the test sample, i.e. the amount of ESM-1 and optionally the amount of natriuretic peptides, compared to the amount of biomarkers in the reference sample. A statistically significant increase is indicative of a subject's response to the therapy. If so, the treatment is effective. Also preferably, a decrease, more preferably a significant decrease, most preferably a statistically significant decrease in the amount of the biomarker in the test sample compared to the amount of the biomarker in the reference sample is non-responsive to treatment. It is an indicator of a subject. In this case the therapy is ineffective.

A subject is considered responsive to a therapy if the therapy reduces the subject's risk of recurrence of atrial fibrillation. A subject is considered unresponsive to a therapy if the therapy does not reduce the subject's risk of recurrence of atrial fibrillation.

In one aspect, if the subject does not respond to the therapy, the intensity of the therapy is increased. Additionally, if the subject responds to therapy, the intensity of the therapy is reduced. For example, the intensity of therapy can be increased by increasing the dose of drug administered. For example, the intensity of therapy can be reduced by reducing the dose of drug administered.

In another aspect, the assessment of atrial fibrillation therapy is guidance for atrial fibrillation therapy. The term "guidance" as used herein preferably refers to adjusting the intensity of therapy during the course of therapy, e.g. dose of oral anticoagulants, preferably based on determination of biomarkers (e.g. ESM-1). relating to increasing or decreasing.

In a further preferred embodiment, the assessment of atrial fibrillation therapy is stratification of atrial fibrillation therapy. Thus, it will be identified which of the subjects are eligible for a given atrial fibrillation therapy. The term "stratification" as used herein preferably relates to selecting an appropriate treatment based on a particular risk, identified molecular pathways and/or expected efficacy of a particular drug or procedure. . Patients with minimal or no symptoms associated with arrhythmia, depending on the risk detected, would be eligible controls for control of heart rate, cardioversion or ablation. Such patients would otherwise receive only antithrombotic therapy.

The terms "significant" and "statistically significant" are known to those skilled in the art. Thus, whether an increase or decrease is significant or statistically significant can be determined without undue effort by one skilled in the art using various well-known statistical evaluation tools. For example, a significant increase or decrease is an increase or decrease of at least 10%, especially at least 20%.

The invention further relates to a method of aiding the assessment of atrial fibrillation, said method comprising
a) obtaining a sample from a subject as referred to herein in connection with a method of assessing atrial fibrillation;
b) determining the amount of the biomarker ESM-1 and optionally the amount of natriuretic peptides in said sample; and
c) providing information regarding said determined amount of biomarker ESM-1 and optionally said determined amount of natriuretic peptides to a subject's attending physician to assist in the assessment of atrial fibrillation in said subject; include.

The attending physician should be the physician who requested the biomarker determination. The methods described above will assist the attending physician in assessing atrial fibrillation. Thus, the method does not encompass diagnosis, prognosis, monitoring, differentiation, identification as referred to above in relation to methods of assessing atrial fibrillation.

Step a) of the above method of obtaining a sample does not involve removing (collecting) the sample from the subject. Preferably, the sample is obtained by receiving a sample from said subject. Thus the sample can have been shipped.

In one aspect, the method is a method of aiding in stroke prediction, comprising:
a) obtaining a sample from a subject as referred to herein in relation to a method of assessing atrial fibrillation, in particular in relation to a method of predicting atrial fibrillation;
b) determining the amount of the biomarker ESM-1 and optionally the amount of natriuretic peptides in said sample; and
c) providing information about said determined biomarker amount and optionally said determined natriuretic peptide amount to the subject's attending physician, thereby aiding in the prediction of stroke.

The present invention further provides
a) providing a test for the biomarker ESM-1 and optionally a test for natriuretic peptides, and
b) providing instructions for utilizing test results obtained or obtained by said test in the assessment of atrial fibrillation.

The purpose of the method is preferably an aid in the assessment of atrial fibrillation.

The instructions will include a protocol for performing the atrial fibrillation assessment method as described above. Further, the directive will contain at least one numerical value for the reference amount of ESM-1 and optionally at least one value for the reference amount of natriuretic peptides.

A "test" is preferably a kit adapted to carry out a method of assessing atrial fibrillation. The term "kit" is explained below. For example, the kit comprises at least one detection agent for the biomarker ESM-1 and optionally at least one detection agent for natriuretic peptides. Detection agents for the two biomarkers can be provided in a single kit or in two separate kits.

The test result obtained or obtained by said test is a value for the biomarker amount.

In one aspect, step b) comprises providing instructions for utilizing test results obtained or obtained by said testing in the prediction of stroke (as described herein).

Further, the present invention provides for assessing atrial fibrillation in a sample of a subject,
i) the biomarker ESM-1 and optionally natriuretic peptides, and/or
ii) to the use of at least one detection agent that specifically binds to ESM-1 and optionally at least one detection agent that specifically binds to natriuretic peptides.

In one aspect, the invention provides, in combination with a clinical stroke risk score, for assessing stroke in a sample of a subject,
i) biomarker ESM-1 and/or
ii) with the use of at least one detection agent that specifically binds to ESM-1.

The present invention further provides, in a sample of subjects, in combination with a clinical stroke risk score for predicting a subject's risk of suffering a stroke,
i) biomarker ESM-1 and/or
ii) with the use of at least one detection agent that specifically binds to ESM-1.

The present invention also provides the biomarker ESM-1 and/or ii) at least one biomarker that specifically binds to ESM-1 to improve the accuracy of predicting clinical stroke risk score in a sample of a subject. Concerning the use of detection agents.

The terms "sample", "subject", "detection agent", "ESM-1", "specifically binds", "atrial fibrillation" and "assessing atrial fibrillation" referred to in connection with the above uses "does" has already been defined in relation to methods of assessing atrial fibrillation. Therefore, the definitions and explanations apply accordingly.

Preferably said use is an in vitro use. Further, the detection agent is preferably an antibody (or antigen-binding fragment thereof), such as a monoclonal antibody.

Furthermore, in the studies of the present invention, it was demonstrated that the determination of the amount of ESM-1 in a sample from a subject allows the diagnosis of heart failure and the structural or functional abnormalities of the heart associated with heart failure. Accordingly, the present invention also relates to methods of diagnosing heart failure and/or at least one structural or functional abnormality associated with heart failure based on the biomarker ESM-1.

The definitions given above apply mutatis mutandis below (except where stated otherwise).

Accordingly, the present invention provides a method of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure in a subject, comprising:
a) determining the amount of ESM-1 in the subject's sample, and
b) comparing said amount of ESM-1 to a reference amount, whereby heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure can be diagnosed.

As used herein, the term "diagnosis" assesses whether the subject referred to in accordance with the methods of the present invention is suffering from heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. means that In one aspect, the subject is diagnosed with heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. In another aspect, the subject is diagnosed as not suffering from heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure (heart failure and/or at least one structural and/or functional abnormality of the heart). excluded from functional abnormalities).

The actual diagnosis of whether a subject is suffering from heart failure and/or said at least one abnormality may involve additional steps such as confirmation of the diagnosis. Thus, the diagnosis of heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure is understood to aid in the diagnosis of heart failure and/or at least one structural or functional abnormality. Accordingly, the term "diagnosing" in the context of the present invention also encompasses assisting a physician in assessing whether a subject is suffering from heart failure and/or said at least one abnormality.

The term "heart failure" (abbreviated as "HF") is well known to those skilled in the art. As used herein, the term preferably relates to systolic and/or diastolic dysfunction of the heart that accompanies overt symptoms of heart failure as known to those skilled in the art. Preferably, the heart failure referred to herein is also chronic heart failure. Heart failure according to the present invention includes overt and/or progressive heart failure. In overt heart failure, the subject exhibits symptoms of heart failure as known to those of skill in the art.

In one aspect of the invention, the term "heart failure" refers to heart failure with reduced left ventricular ejection fraction (HFrEF).

In another aspect of the invention, the term "heart failure" refers to heart failure with preserved left ventricular ejection fraction (HFpEF).

HF can be classified into various degrees of severity.

According to the NYHA (New York Heart Association) classification, heart failure patients are classified into those belonging to NYHA I, II, III and IV. Heart failure patients already experience structural and functional changes in their pericardium, myocardium, coronary circulation or heart valves. Patients cannot fully recover their health and require medical attention. NYHA grade I patients have no obvious signs of cardiovascular disease but already have objective evidence of functional impairment. Patients with NYHA grade II have slight limitations in physical activity. Patients with NYHA grade III have marked limitations in physical activity. NYHA grade IV patients are unable to perform any physical activity without discomfort. Those patients present with symptoms of cardiac insufficiency at rest.

This functional classification has been supplemented by recent classifications by the American College of Cardiology and the American Heart Association (see J. Am. Coll. Cardiol. 2001; 38; 2101-2113 (revised 2005); J. Am. (See Coll. Cardiol. 2005; 46; e1-e82). Four stages A, B, C, D were defined. Stages A and B are believed to help identify patients early, before they develop 'true' HF but not HF. Stage A and B patients are best defined as those with risk factors for developing HF. For example, patients with coronary artery disease, hypertension or diabetes who have not yet manifested impaired left ventricular (LV) function, hypertrophy or geometric ventricular distortion are assigned as stage A, while asymptomatic but Patients with coronary artery disease, hypertension or diabetes who manifest impaired left ventricular (LV) function, hypertrophy or geometric ventricular distortion are assigned as stage B. Stage C then refers to patients with current or past symptoms of HF associated with underlying organic heart disease, and Stage D represents patients with truly refractory HF.

As used herein, the term "heart failure" preferably includes stages A, B, C and D of the ACC/AHA classification described above. The term also includes NYHA degrees I, II, III and IV. Thus, the subject may or may not exhibit typical symptoms of heart failure.

In a preferred embodiment, the term "heart failure" refers to heart failure stage A or in particular heart failure stage B according to the ACC/AHA classification described above. Identification of these early stages, especially stage A, is advantageous as treatment can be initiated before irreversible damage occurs.

At least one structural or functional abnormality of the heart associated with heart failure preferably comprises functional and/or structural damage of the myocardium, epicardium, valves or coronary circulation; reduced pumping or filling capacity, often contraction or Left ventricular geometric alterations; hypertension with geometric ventricular distortion; left ventricular hypertrophy; left ventricular structural alterations with or without left ventricular hypertrophy; increased septal diameter; , concentric myocardial hypertrophy, eccentric myocardial hypertrophy, left ventricular dysfunction, diastolic left ventricular dysfunction.

In one aspect of the invention, the cardiac structural or functional abnormality associated with heart failure is left ventricular hypertrophy.

As described herein, the biomarker ESM-1 can be elevated in various diseases and disorders other than atrial fibrillation. In one aspect of the methods of the invention described above, it is envisioned that the subject does not have such a disease or disorder. For example, the subject will not have chronic kidney disease, diabetes, cancer, coronary artery disease, hypertension, and/or renal failure requiring dialysis. Further, it is assumed that the subject has no history of stroke.

The subject tested according to the method of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure preferably does not suffer from atrial fibrillation. However, it is also envisioned that the subject is suffering from atrial fibrillation. The term "atrial fibrillation" is defined in relation to methods of assessing heart failure.

Preferably, the subject to be tested is suspected of suffering from heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure.

The term "reference amount" has already been defined in relation to methods of assessing atrial fibrillation. The reference amount applied to the method of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure can theoretically be determined as described above.

Preferably, the amount of ESM-1 in the subject's sample is increased compared to the reference amount in a subject suffering from heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. and/or an amount of ESM-1 in a sample of the subject that is reduced relative to a reference amount (or amounts) is indicative of heart failure and/or at least one structure of the heart associated with heart failure It is an indicator of subjects who do not suffer from physical or functional abnormalities.

In one embodiment of the method of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure in a subject, step a) further comprises determining the amount of natriuretic peptides in a sample of the subject. In step b) of the method, the determined amount of this marker is compared with a reference amount.

Preferably, the ratio of NT-proBNP/ESM-1 is an index that distinguishes patients with atrial fibrillation from heart failure, as described below.

Further, the present invention provides a method for diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure in a sample of a subject,
i) the biomarker ESM-1 and optionally natriuretic peptides, and/or
ii) relates to the use of at least one detection agent that specifically binds to ESM-1 and optionally at least one detection agent that specifically binds to natriuretic peptides.

The invention further provides a method of assisting in the diagnosis of heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure in a subject, comprising:
a) obtaining a sample from a subject as referred to herein in connection with a method of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure in a subject;
b) determining the amount of the biomarker ESM-1 and optionally the amount of natriuretic peptides in said sample, and
c) providing information about said determined amount of biomarker ESM-1 and optionally said determined amount of natriuretic peptides to said subject's attending physician, whereby heart failure and/or heart failure associated with heart failure in said subject; aiding in diagnosing at least one structural or functional abnormality of

The attending physician is the physician who requested the biomarker determination. The methods described above will assist the attending physician in assessing atrial fibrillation. Thus, the method does not encompass diagnosing, prognosticating, monitoring, distinguishing, identifying as referred to above in connection with methods of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. .

Step a) of said method of obtaining a sample does not involve drawing the sample from the subject. Preferably, the sample is obtained by receiving a sample from said subject. Thus, the sample would have been shipped.

The present invention further provides
a) providing a test for the biomarker ESM-1 and optionally a test for natriuretic peptides, and
b) providing instructions for utilizing test results obtained or obtained by said test in a method for diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure; Regarding the method of containing.

The purpose of the above method is preferably to aid in the diagnosis of heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure.

The instructions will include a protocol for performing a method for diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure as described above. Further, the directive will contain at least one numerical value for a reference amount of ESM-1 and optionally at least one numerical value for a reference amount of natriuretic peptides.

A "test" is preferably a kit adapted to carry out a method of diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. The term "kit" is explained below. For example, the kit comprises at least one detection agent for the biomarker ESM-1 and optionally at least one detection agent for natriuretic peptides. Detection agents for the two biomarkers can be provided in a single kit or in two separate kits.

The test result obtained or obtained by said test is a value for said biomarker amount.

The invention also relates to kits. Preferably, said kit is adapted to perform the method of the invention, i.e. a method of assessing atrial fibrillation or diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. It is a kit that does. The kit contains at least one agent that specifically binds ESM-1. In preferred embodiments, the kit will further comprise an agent that specifically binds to a natriuretic peptide (eg NT-proBNP). Optionally, the kit includes instructions for carrying out the method.

As used herein, the term "kit" refers to a collection of the above-described components, either provided separately or provided in a single container. The container also contains instructions for practicing the method of the invention. Those instructions may be in manual form, or they may perform calculations and comparisons relating to the method of the invention, and establish an evaluation or diagnosis based thereon when implemented on a computer or on a data processing apparatus. may be provided by computer program code. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (eg compact disc; CD) or may be provided directly on a computer or data processing device. In addition, the kit can preferably contain a standard amount of the biomarker ESM-1 for calibration. In a preferred embodiment, the kit further comprises standard amounts of natriuretic peptides for calibration.

In one aspect, the kit is used to assess atrial fibrillation in vitro.

In another aspect, the kit is used to diagnose heart failure and/or at least one structural or functional abnormality associated with heart failure in vitro.

Calculating the ESM-1 to Natriuretic Peptide Ratio Atrial fibrillation and heart failure share common risk factors that predispose them to diseases such as coronary artery disease. It is also an established finding that patients with heart failure often develop atrial fibrillation (or vice versa).

The Examples show that ESM-1 is a marker for both atrial fibrillation and heart failure (see Figure 6). Biomarkers can therefore be used to diagnose heart failure and assess atrial fibrillation. For example, the presence of heart failure or atrial fibrillation in a subject can be ruled out based on determination of ESM-1 (as described above).

If the level of ESM-1 is increased compared to the reference amount, the subject can theoretically be suffering from atrial fibrillation or heart failure or both. In this case, it would be advantageous to distinguish between heart failure and atrial fibrillation. Such discrimination can be performed by calculating the ratio of the amount of natriuretic peptide (eg, NT-proBNP or BNP) in a sample from a subject to the amount of ESM-1 in a sample from the same subject.

Although the amount of natriuretic peptides is increased in samples from patients with atrial fibrillation, generally the amount of natriuretic peptides is much higher in samples from heart failure patients than from atrial fibrillation patients. For example, in samples from subjects with atrial fibrillation, levels of up to 3000 pg/mL of NT-proBNP were observed in the studies underlying the present invention, whereas in samples from subjects with heart failure not observed. In contrast, subjects with heart failure had NT-proBNP levels up to 15000 pg/mL.

Having increased levels of ESM-1 due to differences in levels of natriuretic peptides, particularly NT-proBNP, between subjects with atrial fibrillation and heart failure It is possible to distinguish between heart failure and atrial fibrillation in a subject (ie, a subject with an increased amount of ESM-1 in the sample compared to a reference amount).

Preferably, the diagnosis of heart failure or atrial fibrillation is in step a) of said method of assessing atrial fibrillation or in step a) of a method of diagnosing heart failure and/or at least one structural or functional abnormality associated with heart failure. is further supported or confirmed by performing the additional step of calculating the ratio of the amount of natriuretic peptide to the amount of ESM-1. In an additional step, the calculated ratio is compared to a reference ratio (ratio of natriuretic peptide amount to ESM-1 amount). Said reference ratio will allow discrimination between heart failure and atrial fibrillation (especially in subjects with increased amounts of ESM-1).

Accordingly, methods of assessing atrial fibrillation (e.g., methods of diagnosing atrial fibrillation) and methods of diagnosing heart failure and/or at least one structural or functional abnormality associated with heart failure are provided below:
- calculating the ratio between the amount of natriuretic peptide determined in step a) and the amount of ESM-1 determined in step a); and - comparing said calculated ratio to a reference ratio. .
The calculating step is preferably step c) and the comparing step is preferably step d).

If steps c) and d) are performed, in step a) of the method of assessing atrial fibrillation and of diagnosing heart failure, the amount of biomarker ESM-1 and the amount of natriuretic peptides are determined. However, in this case it is not necessary to compare the amount of natriuretic peptide to a reference amount in step b). The method of the invention can be performed with or without this comparison in step b).

Thereby a diagnosis of atrial fibrillation or a diagnosis of heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure is verified, confirmed or corroborated. In particular said ratio reduces the number of false positives.

The calculating and comparing steps described above are preferably performed in a subject in whom the amount of ESM-1 in the sample determined in step a) is increased compared to the reference amount.

With respect to methods of assessing atrial fibrillation, in particular with respect to diagnosing atrial fibrillation, the following applies: Preferably, a decreased ratio compared to a reference ratio is indicative of a subject suffering from atrial fibrillation. . Such a ratio thus confirms the diagnosis of atrial fibrillation based on steps a) and b). An increased ratio compared to the reference ratio is indicative of heart failure. Such a ratio thus indicates that the subject is likely not suffering from atrial fibrillation. Therefore, additional diagnostic measures such as ECG for the diagnosis of atrial fibrillation are recommended or initiated.

As regards the method of diagnosing heart failure, the following applies: Preferably, an increased ratio compared to the reference ratio is indicative of a subject suffering from heart failure. An increased ratio therefore confirms the diagnosis of heart failure based on steps a) and b). A decreased ratio compared to the reference ratio is indicative of a diagnosis that the subject does not have heart failure. Such a ratio thus indicates that the subject does not suffer from atrial fibrillation.

Therefore, the present invention provides a subject comprising the steps of determining the amount of ESM-1 and the amount of NT-proBNP in a sample from a subject, and comparing the amount of ESM-1 and the amount of NT-proBNP as a ratio. It relates to methods of diagnosing both said diseases and detecting said risk factors in the body. A subject suspected of suffering from heart failure will have a higher NT-proBNP/ESM-1 ratio compared to a subject suffering from atrial fibrillation.

All references cited herein are hereby incorporated by reference for their entire disclosure content and the disclosure content specifically mentioned in this specification.

The drawing shows:
Figure 1: Measurement of ESM-1 ELISA in the mapping cohort. Figure 2: ROC curve of ESM-1 in the paroxysmal Afib group of the mapping cohort; AUC = 0.61. Figure 3: ROC curve of ESM-1 in the persistent Afib group of the mapping cohort; AUC = 0.89. Figure 4: Diagnostic value of ESM-1 in the PREDICTOR AFib subpanel. Figure 5: ESM-1 diagnostic value in the PREDICTOR AFib subpanel; AUC = 0.68. Figure 6: ESM-1 in distinguishing between heart failure and atrial fibrillation. Figure 7: ESM-1 in distinguishing heart failure; ROC curve for ESM-1; AUC = 0.81. Figure 8: ESM-1 in distinguishing atrial fibrillation; ROC curve for ESM-1 in the Afib group; AUC=0.98. Figure 9: Weighted Kaplan-Meier survival estimates for the two groups defined by baseline ESM-1 measurements (<461 pg/mL vs. >461 pg/mL).

The invention is illustrated solely by the following examples. The examples should not be construed in any way to limit the scope of the invention.
Example 1: Mapping Test - Atrial Fibrillation Patient Diagnosis Compared Based on Different Circulating ESM-1 Levels

MAPPING studies were relevant for patients undergoing open-heart surgery. Samples were taken before anesthesia and surgery. Patients were electrophysiologically characterized using high-density epicardial mapping using a multi-electrode array (high-density mapping). The study included 14 patients with paroxysmal atrial fibrillation, 16 patients with persistent atrial fibrillation, and 30 controls, who were matched to the best possible extent (with respect to age, sex, comorbidities). ESM-1 was measured in samples for mapping studies. Elevated ESM-1 levels were observed in patients with atrial fibrillation compared to controls. Elevated ESM-1 levels were observed in patients with paroxysmal atrial fibrillation versus controls and in patients with persistent atrial fibrillation versus matched controls.

Additionally, the biomarker NT-proBNP was measured in samples from the mapping cohort. Interestingly, it was demonstrated that combined measurement of ESM-1 and NT-proBNP could increase the AUC to 0.91 for discrimination between sustained AF versus SR (sinus rhythm).

Example 2: Predictor Cohort - Screening and Identification of Atrial Fibrillation Patients Specifically in the Elderly General Population

The predictor study was a population-based study in elderly (≧65 years) apparently healthy subjects (n=2001). Participants were referred to a cardiology center for clinical trials and comprehensive Doppler echocardiography and electrocardiography. Patients primarily suffered from stage B heart failure. However, some patients had stage A or C heart failure. The atrial fibrillation subcohort (subgroup) included 40 patients with an ongoing episode of atrial fibrillation during the study and 116 matched controls. Thirty-three patients exhibited risk factors for HF stage A, 11 of whom presented with ongoing atrial fibrillation at the time of sampling. Eighty patients presented with structural heart disease, HF stage B, 21 of whom presented with ongoing atrial fibrillation at the time of sampling. Thirty-seven patients had HF stage C, 6 of whom presented with ongoing atrial fibrillation at the time of sampling.

ESM-1 and NT-proBNP were measured in an atrial fibrillation subcohort selected from the PREDICTOR study. Elevated circulating ESM-1 levels were observed in samples from patients with ongoing atrial fibrillation relative to controls. Slightly elevated circulating ESM-1 levels were observed in patients with structural and functional myocardial abnormalities (Stage C HF) compared to controls. Elevated circulating NT-proBNP levels of approximately 120 pg/mL or greater were observed in patients with ongoing atrial fibrillation compared to controls. Elevated circulating NT-proBNP levels of 120 pg/mL or greater were observed in patients with stage C HF compared to controls.

Example 3 Heart Failure Panel A heart failure panel included 60 patients with chronic heart failure. Heart failure was diagnosed in patients with typical signs and symptoms and objective evidence of cardiac structural or functional abnormalities at rest, according to ESC guideline criteria. Patients aged 18 to 80 who had ischemic or dilated cardiomyopathy or severe valvular disease and were able to sign consent forms were included in the study. Patients with acute myocardial infarction, pulmonary obstruction or stroke within the last 6 months, as well as severe pulmonary hypertension and end-stage renal disease were excluded. Patients mostly had heart failure stages NYHA II-IV.

The atrial fibrillation cohort included 10 elderly patients (>60 years), including those diagnosed with hypertension and those with heart disease.

A healthy control cohort included 56 subjects. I checked my health and got an ECG and an echocardiogram. Participants with any abnormalities were excluded. Elevated ESM-1 levels were observed in heart failure patients compared to controls.

Example 4: GISSI AF test
The GISSI-AF study requires at least 2 episodes of ECG-confirmed symptomatic atrial fibrillation within the last 6 months or a successful electrical A double-blind, randomized, placebo-controlled, multicenter trial enrolling 1442 patients in sinus rhythm with a history of atrial fibrillation documented by pharmacological defibrillation.

The theoretical principles, design and results of this study have been described in detail previously (see: Disertori M. et al., J Cardiovasc Med. 2006; 7:29-38; see: Staszewsky L. et al., Cardiovasc Drugs Ther 2015; 29:551). -561) and previously published under Clinical Trials.gov (identifier NCT00376272).

All patients were >48 years of age and had received stable treatment for atrial fibrillation and cardiovascular disease for at least 1 month prior to enrollment. Previously prescribed ACE inhibitors, beta-blockers and amiodarone were allowed to continue. Patients were randomized to receive valsartan or placebo.

The primary endpoints of the main study were (a) time to first recurrence of atrial fibrillation and (b) multiple recurrences of atrial fibrillation during the 1-year observation period for the angiotensin type II type 1 receptor blocker valsartan. was to assess the effect on the proportion of patients with the onset of The biomarker substudy (N=382) included 203 subjects with recurrent atrial fibrillation and 179 controls without recurrent atrial fibrillation during the 12-month follow-up period (Masson S et al. Heart. 2010;96:1909-14; Latini R et al., J Intern Med. 2011; 269:160-71). Blood samples were collected at randomization and after 6 and 12 months of follow-up.

The biomarker ESM-1 was measured on samples from the biomarker substudy. Elevated ESM-1 levels were observed in patients with atrial fibrillation compared to controls (AUC 0.61).

Example 5: Biomarker Measurement ESM-1 was measured in a commercial microtiter plate assay for endothelial cell-specific molecule 1 (ESM-1); ELISA kit from Abnova (Taiwan).

Example 6: Stroke Predictive Analysis Approach The ability of circulating ESM-1 to predict the risk of developing stroke was evaluated in a prospective multicenter registry of patients with documented atrial fibrillation (Conen D., Forum Med Suisse 2012; 12:860-862). ESM-1 was measured in EDTA plasma using a stratified case-cohort study design as described by Borgan et al. (Borgan O. et al. Lifetime Data Analysis 2000; 6: 39-58).

Persistent and paroxysmal cases were predominantly in sinus rhythm and persistent cases were in atrial fibrillation during blood collection.

The sample material was EDTA plasma.
Two matched controls were selected for each of the 70 patients who experienced a stroke during the follow-up period ("event"). Controls were matched based on demographic and clinical information of age, sex, history of hypertension, type of atrial fibrillation, and history of heart failure (CHF history).

ESM-1 results were obtained for 69 AF patients with events (ie, stroke) and 138 AF patients without events. There were a total of 207 patients. At the time of blood sampling, 86 of 207 patients were diagnosed with paroxysmal/persistent atrial fibrillation, 50 of 207 had persistent atrial fibrillation, and 71 of 207 had persistent AF. diagnosed as having

The maximum observation period (follow-up period) for those 207 subjects was 42%, 24% and 34%, respectively, with the majority of paroxysmal AF patients in sinus rhythm at the time of blood draw. Stroke follow-up was 2615 days. Median follow-up was 6 years, or up to 1450 days.

A proportional hazards model for the outcome stroke was used to quantify the univariate prognostic value of ESM-1. The univariate prognostic value of ESM-1 was assessed by two different inputs of prognostic information provided by ESM-1. The first proportional hazards model included ESM-1 binarized at the median (461 pg/mL), thus reducing the risk of patients with ESM-1 below the median to those with ESM-1 above the median. A comparison against patients with 1 was included. A second proportional hazards model included the original ESM-1 levels but transformed to the log2 scale. Log2 transformation was performed to allow better model testing. A weighted proportional hazards model was used because the estimates from the simple proportional hazards model for the case-control (case-control) cohort are biased (due to changes in the ratio of cases to controls). Weights are based on each patient's inverse probability of being selected for the case-control cohort as described by Mark et al. (Mark SD et al., J Am Statistical Assoc 2006: 101: 460-471).

A weighted version of the Kaplan-Meier plot was used to obtain estimates for the two groups of absolute survival rats based on binarized baseline ESM-1 measurements (<461 pg/mL vs. ≧461 pg/mL). was prepared as described in Mark et al. (Mark SD et al. J Am Statistical Assoc 2006:101: 460-471).

To assess whether the prognostic value of ESM-1 is a known clinical and demographic risk factor, age, sex, history of CHF, history of hypertension, history of stroke/TIA/thromboembolism, history of vascular disease A weighted proportional Cox model was calculated that included variables such as age and diabetes history.

To assess the ability of ESM-1 to improve the current risk score for stroke prognosis, we calculated the _{CHA2DS2 - VASc} score and ESM-1 (after log2 transformation) in a weighted proportional hazards model including CHADS2, respectively. .

The c-index of the CHA2DS2 _{- VASc} score was compared to the c-index of this model. Calculation of the c-index in the case-cohort setting used a weighted version of the c-index as proposed by Ganna et al.

Results Table 1 shows the results of two univariate weighted proportional hazards models containing ESM-1 after binarization or log2 transformation. The association between the risk of experiencing a stroke and baseline levels of ESM-1 is highly significant in both models. Patients with baseline ESM-1 ≥ 461 pg/mL had a 2.78-fold higher risk of stroke onset of binarized ESM-1 compared to patients with baseline ESM-1 < 461 pg/mL Imply.

Results from a proportional hazards model that includes ESM-1 as a Log2-transformed linear risk predictor suggest that log2-transformed ESM-1 values are proportional to the risk of developing stroke. A hazard ratio of 2.92 can be interpreted in such a way that a 2-fold increase in ESM-1 is associated with a 2.96 increase in stroke risk.

Table 1. Univariate weighted proportional hazards model results including ESM-1 after binarization and log2 transformation

FIG. 9 shows weighted Kaplan-Meier curves for the two patient groups with baseline ESM-1 measurements <461 pg/mL vs. ≧461 pg/mL. As is readily apparent, the two groups of patients have significantly different risks of developing stroke.

Table 2 shows the results of a proportional hazards model including ESM-1 (after log2 transformation) combined with clinical and demographic variables. The results clearly demonstrate that the prognostic effect of ESM-1 remains stable when adjusted for the prognostic effect of relevant clinical and demographic variables.

Table 2: Multivariate proportional hazards model including ESM-1 and related clinical and demographic variables

Table 3 shows the results of a weighted proportional hazards model combining CHADS ₂ scores with ESM-1 (after log2 transformation). In this model as well, ESM-1 can add prognostic information to CHADS ₂ scores.

Table 3: Weighted proportional hazards model combining CHADS ₂ scores with ESM-1 (after log2 transformation)

Table 4 shows the results of a weighted proportional hazards model combining CHA2DS2 _{- VASc} scores with ESM-1 (after log2 transformation). Also in this model, ESM-1 can add prognostic information to the CHA ₂ DS ₂ -VASc score.

Table 4: Weighted proportional hazards model combining CHA2DS2 _{- VASc} scores with ESM-1 (after log2 transformation)

Table 5 shows the CHADS ₂ and CHA2DS2 _- VASc scores in case-cohort screening (N=207) and _{CHADS 2 score} in case-cohort screening of ESM-1 alone in the full BEAT-AF test (N=1537). and CHA ₂ DS ₂ -VASc scores combined with ESM-1 (log2) weighted proportional hazards model, respectively. As expected, the c-indexes of the CHADS ₂ and CHA ₂ DS ₂ -VASc scores for the full cohort differ somewhat from those for case-cohort screening due to random sampling of the case-control cohort. In the case-control cohort, the addition of ESM-1 to the CHA2DS2 _{- VASc} score improved the c-index by 0.057, which can be considered a clinically meaningful improvement in risk prediction. For the CHADS ₂ score, the c-index improvement is 0.064.

Improvement is observed in the prognostic value of persistent atrial fibrillation. Strong improvement is seen in patients with paroxysmal atrial fibrillation. On the other hand, the strongest improvement is seen in the group of patients with persistent atrial fibrillation.

Table 5: ESM-1, CHADS ₂ and CHA2DS2 _{- VASc} scores, and C-index for ESM-1 and their combinations.

Uses The results provided in this example demonstrate that ESM-1, alone or in combination, can be used to predict future stroke risk in new patients with clinical scores (e.g., CHADS ₂ and CHA ₂ DS ₂ -VASc) can be used in several ways to significantly improve DS 2 -VASc). For new patients, ESM-1 can be measured and compared to a defined cutoff (eg, 461 pg/mL). If a new patient's measurements are above a predefined cutoff, the patient is considered to be at high risk of having experienced a stroke and appropriate clinical measurements can be initiated.

It is also possible to define more than two risk groups based on increased cut offsets. Patients will then be assigned to one of the risk groups based on their ESM-1 measurements. The risk of developing stroke differs across different risk groups.

Alternatively, ESM-1 results could be directly converted to continuous risk scores based on an appropriate predetermined conversion function.

Moreover, ESM-1 values can be used in combination with clinical and demographic variables (eg, _{CHA2DS2 -} VAS score), thereby improving the accuracy of risk prediction.

For new patients, the risk score value is evaluated in combination with the ESM-1 value (potentially after log2 transformation) measured in an appropriate manner, e.g. with the risk score result and the appropriate defined weight It will be evaluated by creating a weighted sum with the ESM-1 value.

Claims (31)

A method of providing an index for evaluating atrial fibrillation in a subject, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount, thereby providing an index for assessing atrial fibrillation. 2. The method of claim 1, wherein step a) comprises determining the amount of natriuretic peptide and step b) comprises comparing said amount of natriuretic peptide to a reference amount. 3. The method of claim 1 or 2, wherein said subject is suspected of having atrial fibrillation and said assessment of atrial fibrillation is a diagnosis of atrial fibrillation. An increased amount of ESM-1 and/or natriuretic peptide in a sample of a subject compared to said reference amount is indicative of a subject suffering from atrial fibrillation and/or compared to said reference amount 3. The method of claim 2 , wherein the amount of ESM-1 and/or natriuretic peptide in the subject's sample decreased by 100% is indicative of a subject not suffering from atrial fibrillation. 5. The method of any one of claims 1-4, wherein the amount of ESM-1 in the subject's sample is increased compared to a reference amount. The amount of ESM-1 and the amount of natriuretic peptide are determined in step a), and the method c) determines the amount of natriuretic peptide determined in step a) relative to the amount of ESM-1 determined in step a). 6. The method of claim 5, comprising the additional steps of calculating a ratio and comparing said calculated ratio to a reference ratio. 7. The method of claim 6, wherein a ratio that is reduced compared to the reference ratio is indicative of a subject suffering from atrial fibrillation. the subject is suffering from atrial fibrillation and the atrial fibrillation assessment is the distinction between paroxysmal atrial fibrillation and sustained atrial fibrillation, wherein the subject has increased compared to the reference amount the amount of ESM-1 in the sample is indicative of a subject suffering from persistent atrial fibrillation and/or the amount of ESM-1 in the sample of the subject is decreased compared to a reference amount 2. The method of claim 1, which is indicative of a subject suffering from paroxysmal atrial fibrillation. said subject is suffering from atrial fibrillation and said assessment of atrial fibrillation is a prediction of risk of an adverse event associated with atrial fibrillation, wherein said adverse event is selected from recurrence of atrial fibrillation and stroke 2. The method of claim 1, wherein: an increased amount of ESM-1 in a subject's sample compared to said reference amount is indicative of a subject at risk of developing an adverse event associated with atrial fibrillation and/or compared to said reference amount 10. The method of claim 9, wherein an amount of ESM-1 in the subject's sample that is decreased by 100% is indicative of the subject not at risk of developing an adverse event associated with atrial fibrillation. 3. The method of claim 1, wherein said assessment of atrial fibrillation is identification of a subject to undergo electrocardiography (ECG). 2. The method of claim 1, wherein the subject has atrial fibrillation and the assessment of atrial fibrillation is assessment of a therapy for atrial fibrillation. 1. A method of providing an indication for diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure in a subject, comprising:
a) determining the amount of ESM-1 in a sample from the subject;
b) comparing said amount of ESM-1 to a reference amount, thereby providing an indication for diagnosing heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure. in a sample of a subject a) to assess atrial fibrillation, or b) to diagnose heart failure and/or at least one structural or functional abnormality of the heart associated with heart failure or for the prediction of stroke;
i) the biomarker ESM-1, and/or
ii) use of at least one detection agent that specifically binds to ESM-1. 15. Use according to claim 14, wherein a natriuretic peptide and/or at least one detection agent that specifically binds to a natriuretic peptide is used. A method of aiding the assessment of atrial fibrillation, comprising:
a ) determining the amount of the biomarker ESM-1 in a sample taken from the subject; and
b ) providing information regarding said determined biomarker ESM-1 amount to said subject's attending physician, thereby assisting in the assessment of atrial fibrillation in said subject. wherein said step b) comprises determining the amount of natriuretic peptides in said sample, said step c) providing information regarding said determined amount of natriuretic peptides to said subject's attending physician, thereby 17. The method of claim 16, comprising assisting in the assessment of atrial fibrillation in said subject. A method of aiding the assessment of atrial fibrillation, comprising:
a) providing a test for the biomarker ESM-1, and
b) providing instructions for utilizing test results obtained or obtained by said test in the assessment of atrial fibrillation. 19. The method of claim 18, wherein step a) comprises providing a test for natriuretic peptides. A method of providing an index for predicting stroke risk in a subject, comprising:
a) determining the amount of ESM-1 in a sample of said subject; and
b) comparing said amount of ESM-1 to a reference amount, thereby providing an index for predicting the risk of stroke. 21. The method of claim 20, wherein said stroke is cardioembolic stroke. 22. The method of claim 20 or 21, wherein said stroke is associated with atrial fibrillation. A method of improving the accuracy of predicting a subject's clinical stroke risk score, comprising:
c) determining the amount of ESM-1 in a sample from a subject with a known clinical stroke risk score; and
a) combining said ESM-1 amount with said clinical stroke risk score, thereby improving the predictive accuracy of said clinical stroke risk score. 24. The method of claim 23, further comprising determining the subject's clinical stroke risk score. A method of providing an index for predicting stroke risk in a subject, comprising:
a) measuring the amount of ESM-1 in a sample from a subject with a known clinical stroke risk score , and
b) combining said ESM-1 amount with said clinical stroke risk score, thereby providing an index for predicting stroke risk of said subject. Use of the biomarker ESM-1 and/or at least one detection agent that specifically binds ESM-1 in a sample from a subject to improve the accuracy of predicting a clinical stroke risk score. combined with a clinical stroke risk score to predict a subject's risk of suffering a stroke in a sample of subjects,
i) biomarker ESM-1 and/or
ii) use of at least one detection agent that specifically binds to ESM-1. The method of any one of claims 23 to 25 or the use of claims 26 or 27, wherein said clinical stroke risk score is a CHA ₂ DS ₂ -VASc score or a CHADS ₂ score. A kit comprising an agent that specifically binds to ESM-1 and an agent that specifically binds to a natriuretic peptide. 30. The kit of claim 29, wherein said natriuretic peptide is NT-proBNP. 31. The kit of claim 29 or 30, wherein said agent is a monoclonal antibody.

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